Bacterial Cytological Profiling Reveals the Mechanism of Action of Anticancer Metal Complexes

Article abstract of DOI:10.1021/acs.molpharmaceut.8b00407

Target identification and mechanistic studies of cytotoxic agents are challenging processes that are both time-consuming and costly. Here we describe an approach to mechanism of action studies for potential anticancer compounds by utilizing the simple prokaryotic system, E. coli, and we demonstrate its utility with the characterization of a ruthenium polypyridyl complex [Ru(bpy)₂dmbpy²⁺]. Expression of the photoconvertible fluorescent protein Dendra2 facilitated both high throughput studies and single-cell imaging. This allowed for simultaneous ratiometric analysis of inhibition of protein production and phenotypic investigations. The profile of protein production, filament size and population, and nucleoid morphology revealed important differences between inorganic agents that damage DNA vs more selective inhibitors of transcription and translation. Trace metal analysis demonstrated that DNA is the preferred nucleic acid target of the ruthenium complex, but further studies in human cancer cells revealed altered cell signaling pathways compared to the commonly administrated anticancer agent cisplatin. This study demonstrates E. coli can be used to rapidly distinguish between compounds with disparate mechanisms of action and also for more subtle distinctions within in studies in mammalian cells.

Full text of DOI:10.1021/acs.molpharmaceut.8b00407

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Article
Bacterial Cytological Profiling Reveals the
Mechanism of Action of Anticancer Metal Complexes
Yang Sun, David Kayvon Heidary, Zhihui Zhang, Christopher I Richards, and Edith Caroline Glazer
Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00407 • Publication Date (Web): 04 Jun 2018
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Molecular Pharmaceutics
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Bacterial Cytological Profiling Reveals the Mechanism of Action of
Anticancer Metal Complexes
Yang Sun,‡ David K. Heidary,‡ Zhihui Zhang, Christopher I. Richards, Edith C. Glazer*
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Department of Chemistry, University of Kentucky, Lexington, KY 40506, United States
ABSTRACT: Target identification and mechanistic studies of cytotoxic agents are challenging processes that are both time consum-
ing and costly. Here we describe an approach to mechanism of action studies for potential anticancer compounds by utilizing the
simple prokaryotic system, E. coli, and demonstrate its utility with the characterization of a ruthenium polypyridyl complex
[Ru(bpy)2dmbpy2+]. Expression of the photoconvertible fluorescent protein Dendra2 facilitated both high throughput studies and
single cell imaging. This allowed for simultaneous ratiometric analysis of inhibition of protein production and phenotypic investiga-
tions. The profile of protein production, filament size and population, and nucleoid morphology revealed important differences be-
tween inorganic agents that damage DNA vs. more selective inhibitors of transcription and translation. Trace metal analysis demon-
strated that DNA is the preferred nucleic acid target of the ruthenium complex, but further studies in human cancer cells revealed
altered cell signaling pathways compared to the commonly administrated anticancer agent cisplatin. This study demonstrates E. coli
can be used to rapidly distinguish between compounds with disparate mechanisms of action, but more subtle distinctions within in
studies in mammalian cells.
KEYWORDS: Cancer, drug discovery, cisplatin, ruthenium, bacterial cytological profiling
Despite multiple technicological advances, the identification
of the mechanism of action for cytotoxic compounds remains a
time consuming and challenging process. While simple in vitro
systems such as purified enzymes and nucleic acids can provide
key insights, there are undeniable advantages to working in
living cells. Bacteria are intrinsically simpler systems than
eukaryotic cells, with E. coli containing only 4,288 genes,20-21
as opposed to the approximately 30,000 genes found in the
human genome.22-23 Essential processes are homologues
between bacteria and eukaryote, including DNA replication,
transcription, and translation. It is well known that many agents
that are toxic to eukaryotic systems also have antibacterial
activities, such as classical antitumor antibiotics, though many
orthogonal variations do exist between the two.24-25
INTRODUCTION
The fortuitous observation of filamentous growth of E. coli
by Barnett Rosenberg led to the discovery of cisplatin, one of
the most important and widely used chemotherapeutic agents.1-
3
Cisplatin, and its later generation analogues, are essential
components in clinical treatments of ovarian, testicular, small
cell lung, and head and neck cancers.4-6 The administration of
platinum drugs, however, is limited by adverse side-effects,
including nephrotoxicity, neurotoxicity, ototoxicity and other
complications.7-8 Drug resistance (either intrinsic or acquired)
compromises the efficacy of platinum drugs as well.9-11 These
deficiencies have necessitated the development of new
chemotherapeutic agents to overcome such obstacles.
Significant efforts have been applied in the field of medicinal
inorganic chemistry to identify cytotoxic agents that replicate
the efficacy of cisplatin, with the hope of adding to our current
arsenal of chemotherapeutic drugs.12-14 While many of the new
chemical entities show promising efficacy, the understanding of
their biological activities is often incomplete. The very nature
of inorganic agents (with variable charge states, geometries, and
coordination numbers, all of which can be altered by speciation)
adds to the challenge, and can result in polypharmacology.13, 15
As a result, elucidation of the biological effects of potential
medicinal inorganic agents has lagged far behind chemical
innovation. For example, oxaliplatin, which has been in clinical
use for over twenty years, was recently reported to induce
ribosome biogenesis stress,16 rather than the previously
accepted mechanism involving DNA damage similar to
cisplatin. Organic or inorganic agents developed through target-
based drug discovery avoid some of these pitfalls, but undesired
off-target effects are prevalent for these systems as well. Thus,
mechanistic studies are necessary even for compounds designed
to inhibit single, well-validated targets.17-19
Rosenberg's classical experiment illustrated that a simple
prokaryotic system could be employed to discover anticancer
agents. Recently, other groups, including those of Lippard and
Brabec, have utilized E. coli phenotypic assays as qualitative
means to characterize potential anticancer agents, and as with
Scheme 1. Thermal hydrolysis of A) cisplatin and B) the
photochemical hydrolysis of compound 1.
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µM, followed by 3 min of light irradiation (7 J/cm2 blue light (>
400 nm)). The cells were then incubated for 16 hours with the
compounds, and cell growth was determined by measurement
of the optical density at 600 nm using a SpectraMax Multiwell
Plate Reader (Molecular Devices).
HL60 cells were plated in Opti-MEM supplemented with 1%
FBS and 50 U/mL of Penicillin/Streptomycin at 30,000 cells per
well in 96 well flat bottom transparent tissue culture treated
plates (Greiner Bio One). Compounds were dosed from 0 – 300
µM, and incubated for 16 hrs, followed by light irradiation with
7 J/cm2 blue light (> 400 nm) in 30 second pulses for a total
light exposure of 3 minutes. The cells were then incubated for
72 hours, and cell viability determined by conversion of
resazurin to resorufin. Dark controls were run in parallel. The
emission of resorufin was measured on a SpectraFluor Plus
Plate Reader (Tecan).
The data were normalized to the untreated control and fitted
to a sigmoidal dose response model using Prism 6.02 to
determine IC50 values. Minimal inhibitory concentration (MIC)
was fitted to the model published by Lambert et al using Prism
6.02.32
cisplatin, showed a good correlation between activity in the
prokaryotic system and cancer cells.26-28 We also have an
interest in simple biological systems, but our motivation is
instead to utilize them as a tool to investigate mechanistic
details of anticancer agents. Our premise is that compounds
which are found to be active in mammalian cells but not E. coli
can be expected to affect processes or targets absent in the
simpler biological system. Alternatively, compounds which
show similar activities in the two cell types can be deduced to
inhibit processes common to both. Thus, it should be possible
to use E. coli as a first-pass screen to radically reduce the
number of likely biological entities or processes targeted by
cytotoxic agents. Furthermore, E. coli is readily amemnable to
incorporation of genetically encoded reporter systems, allowing
for additional phenotypic analysis to be used to rapidly parse
mechanistic features of active compounds.18, 29 This approach
could greatly expidite mechanism of action studies.
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Here we describe studies that demonstrate that E. coli is an
excellent model for mammalian systems to investigating the
effect of metal complex inhibition of cell growth, and
phenotypic changes consistent with DNA damage.30
A
promising light-activated ruthenium complex developed in our
laboratory31 (compound 1, Scheme 1) was compared to cisplatin,
along with three organic antibiotics. Noteworthy differences
were observed between the inorganic compounds and organic
compounds in the bacterial system; these differences directly
correlate with their different mechanisms of action. Moreover,
differences between compound 1 and cisplatin in mammalian
cells suggest more subtle disparities in their mechanistic
features, which offers the possibility to maintain anticancer
efficacy without experiencing the same resistance profile by
altering the metal center from platinum to ruthenium.
Protein synthesis inhibition
E. coli BL21DE3 cells transformed with pCWori-Dendra2 were
cultured in LB medium to an OD600 of 0.8. Cells were then
resuspended in M63 minimal media and induced with 0.5 mM
IPTG for 3 hours at 37 ℃ with 180 rpm shaking.
Photoconversion of Dendra2 was carried out with a 405 nm
LED flood array (Loctite) with a total light exposure time of 2
minutes. Cells were then plated in 96 well plates at 6 x 107 cells
per well. Green and red emission was measured directly after
photoconversion using a SpectraMax Multiwell Plate Reader
(Molecular Devices) for a baseline evaluation of Dendra2
protein (t = 0 hour). For green emission, an excitation
wavelength of 491 nm and emission wavelength of 538 nm was
used; for red emission, the excitation wavelength was 544 nm
and emission wavelength was 590 nm. Compounds were then
dosed from 0 µM to 300 µM, and compound 1 was activated
with light as described above. The cells were incubated for 16
hours before the green and red emission was measured again for
an evaluation of protein synthesis with compound treatment (t
= 16 hours).
METHODS
E. coli culture maintenance
The Dendra2 gene was cloned into a pCW-ori plasmid modified
to contain an N-terminal 6x histidine tag with multiple
restriction enzyme cloning sites. Escherichia coli BL21(DE3)
competent cells transformed with pCWori plasmid containing
Dendra2 gene (pCWori-Dendra2) were cultured in Luria Broth
(LB) at 37 ℃ with 180 rpm shaking.
Mammalian cell maintenance
The average fluorescence ratio of green/red at t = 0 hour and
t = 16 hours was calculated, the values were normalized, and
the data fitted to a sigmoidal dose response.
Human promyelocytic leukemia HL60 cells were purchased
from ATCC. Dulbecco's modified eagle medium (DMEM),
Iscove's modified Dulbecco's medium (IMDM), Opti-MEM I
reduced serum medium, heat inactivated fetal bovine serum
(FBS), Penicillin/Streptomycin (5,000U/mL), Trypsin-EDTA
(0.5%), Dulbecco's phosphate buffered saline (DPBS), Trypan
Blue Solution (0.4%) were purchased from Life Technologies.
HL60 cells were maintained in IMDM supplemented with 10%
FBS and 50 U/mL of Penicillin/Streptomycin. A549 cells were
maintained in DMEM with the same supplements. Cells were
maintained at 37℃ with 5% CO2.
E. coli filamentous growth
E. coli were cultured as above and plated at 3 x 108 cells per
well in 24 well flat bottom transparent tissue culture treated
plates (Greiner Bio One). IPTG was added at a concentration of
0.5 mM for induction of Dendra2 production. Compound
treatment was then carried out, with cells dosed at the MIC or
10x MIC for each compound and cultured at 37 ℃ with 180
rpm of shaking for 6 and 16 hours before imaging.
Cytotoxicity determination
E. coli cell imaging
E. coli BL21(DE3) cells transformed with pCWori-Dendra2
plasmid were plated in M63 minimal medium at 4 x 106 cells
per well in 96 well flat bottom transparent tissue culture treated
plates (Greiner Bio One). Compounds were dosed from 0 – 300
After compound treatment, E. coli cells were centrifuged at
8000 rpm for 2 minutes, washed twice with PBS, and 3 x 107
cells were resuspended in 1 mL of PBS. The fluorescent dyes
FM4-64 and Hoechst 33342 were added to a final concentration
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analysis, and cells were washed twice with PBS. Total RNA and
of 5 µg/mL and 10 µg/mL respectively. The cells were protected
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from light for 20 minutes, and then 2 µL of cell suspension was
placed on a slide and a cover glass was applied before imaging.
Imaging was carried out on an Olympus IX2-RFAEVA-2
microscope with the following filter settings:
genomic DNA were also isolated, and the nucleic acids, cell
content, and media were prepared for analysis as described
above.
Dendra2 (green), excitation filter: 473/10 nm BrightLine®
single-band bandpass filter, FF01-473/10-25 (Semrock,
Rochester, NY, USA); emission filter: 525/50 nm BrightLine®
single-band bandpass filter, FF03-525/50-25 (Semrock,
Rochester, NY, USA). Dendra2 (red) and FM4-64, excitation
filter: HQ 550/30 (Chroma, Bellows Falls, VT, USA); emission
filter: 664 nm EdgeBasic long-pass edge filter, BLP01-664R-
25 (Semrock, Rochester, NY, USA). Hoechst 33342, excitation
filter: BP 360-390 (Chroma, Bellows Falls, VT, USA);
emission filter, HQ470/30 M (Chroma, Bellows Falls, VT,
USA).
Immunoblotting
HL60 cells were harvested 0, 1, 3, 6, 12, 24, 30 and 48 hours
after treatment, pelleted by centrifugation at 124 x g for 5
minutes, washed twice with DPBS. A549 cells were plated at 2
x 105 cells per well in 6 well flat bottom transparent tissue
culture treated multiwell plates and in the same treatment
condition detailed for HL60 cells. Cells were harvested at 0, 6,
12, 24 and 48 hours after treatment.
All cells were lysed in RIPA buffer supplemented with 5 mM
sodium pyrophosphate (2 x 106 cells/100 µL) for 15 minutes on
ice. The insoluble fraction was removed by centrifugation at
20,817 x g for 10 minutes at 4 °C. The supernatant was collected
and the protein concentration was determined by BCA assay.
20 µg of protein was loaded onto 4-12% bis-tris gels and
followed by transfer to nitrocellulose membranes. After
blocking with 2.5% BSA in DPBS with 0.1% Tween20 (PBST)
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Imaging data was processed and analyzed with ImageJ.
Metal uptake in bacterial cells
E. coli were cultured in M63 minimal medium as discussed
above and dosed with 20 µM compound 1 or cisplatin. Cells
treated with compound 1 were irradiated with 7 J/cm2 blue
filtered light (> 400 nm) for a total of 3 minutes, or were
protected from light. Cells were collected 24 hours after
compound addition by centrifugation at 8000 rpm for 5 minutes.
The culture medium was separated for analysis, and cells were
washed twice with PBS and pelleted. Both cell content and
medium were heated at 110 ℃ for 3 hours with 20% (v/v) HNO3.
Total RNA and genomic DNA were isolated using Qiagen kits.
RNA and DNA samples were digested in HNO3 as described
above. Following sample digestion, the metal content was
analyzed using a Varian AAS with a replicate reading and a
spiked reading.
for
1 hour at room temperature, the membrane was
immunoblotted with the following primary antibodies and
correspondence dilutions.
Cleaved caspase 3, cleaved PARP, p-p53, p21, p-Chk1, p-JNK
and γ-H2AX at 1:1000 dilutions; p53 and p-ERK at 1:500
dilutions; and GAPDH at a 1:2000 dilution in 2.5% BSA
overnight at 4 °C. Immunoblots were washed with PBST for 10
minutes for four times and incubated for 1 hour with secondary
antibodies at a 1:10000 dilution for GAPDH and 1:5000
dilutions for all other antibodies. Detection was carried out with
Clarity Western ECL Substrate and imaged with a ChemiDoc
MP System (Bio-Rad).
Cellular uptake was calculated as follows:
DNA fragmentation
HL60 cells were cultured, and treated as described above. Cells
were harvested at 0, 3, 8, 12, 24 and 30 hours after treatment,
pelleted by centrifugation at 124 x g for 5 minutes, washed
twice with DPBS, and prepared with an apoptotic DNA-ladder
kit as per manufacturer instructions (Rosch). Gel
electrophoresis was carried out using a 1 % agarose gel
containing 0.5 µg/mL ethidium bromide for 90 minutes at 75 V.
Gel imaging was performed with the ChemiDoc MP.
Genomic DNA and total RNA were quantified by measuring
their absorbance at 260 nm. Mass to DNA nucleotide pair
conversion was calculated using the average molecular weight
of DNA nucleotide pairs. The number of DNA nucleotide bases
per metal center was calculated as following.
Mass to RNA nucleotide base conversion was calculated using
the average molecular weight of RNA nucleotide bases.
Number of RNA nucleotide bases per metal center was
calculated as following.
Flow cytometry
HL60 cells were cultured, and treated as detailed previously.
Cells were harvested at 24 hours after treatment, pelleted by
centrifugation at 124 x g for 5 minutes, washed twice with
DPBS. For cell death mechanism analysis, cells were stained 15
minutes with FITC-Annexin V and PI; for cell cycle analysis,
cells were stained 15 minutes with PI only. Cells were analyzed
with a FACSCalibur (Becton-Dickenson). A minimum of
20000 events were measured for each sample.
Metal uptake in HL60 cells
HL60 cells were plated in Opti-MEM supplemented with 1%
FBS and 50 U/mL Penicillin/Streptomycin at a density of 1x
106 cells/mL in 25 cm2 cell culture flasks, and dosed with 20
µM compound 1 or cisplatin. Cells treated with compound 1
were incubated 12 hours protected from light before irradiating
with 7 J/cm2 blue filtered light (> 400 nm) in 30 second pulses
for a total of 3 minutes, or protected from light. Cells were
collected 24 hours after compound addition by centrifugation at
124 x g for 5 minutes. The culture media was separated for
RESULTS
Comparison of Compound Efficacies in E. coli and
Mammalian Cancer Cells.
The capacity of E. coli to serve as a model system for cancer
cells was first evaluated by comparing the relative cytotoxicities
of the metal complexes in the two cell types. The ruthenium
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Table 1. Cytotoxicity values and inhibition of protein production for various compounds in E. coli and HL60 cells.
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E. coli
HL60
Growth Inhibition IC50
(µM)
Dendra2 Production Inhibition
IC50 (µM)
Cytotoxicity IC50
(µM)
MIC (µM)
1 light
6.1 ± 0.8
> 300
2.6 ± 0.4
> 300
77 ± 3
> 300
3.4 ± 0.3
> 300
2.6 ± 0.4
n.d.
1 dark
Cisplatin
Rifampicin
Tetracycline
Nalidixic Acid
4.4 ± 0.5
0.6 ± 0.3
10 ± 1.1
5.2 ± 2.0
2.0 ± 0.1
0.3 ± 0.1
4.8 ± 0.7
2.6 ± 0.4
85 ± 11
2.8 ± 0.3
1.3 ± 0.2
4.6 ± 0.1
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n.d.
n.d.
Table 2. Cellular metal uptake and metal content with different nucleic acids measured by AAS.
E. coli
HL60
Cellular uptakea
DNA nt / mc b
RNA nt / mcc
3800 ± 600
Cellular uptakea
DNA nt / mcb
RNA nt / mcc
5000 ± 700
1 light
10%
2%
2000 ± 200
0.64%
0.11%
0.72%
4800 ± 400
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1 dark
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-
-
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Cisplatin
6%
3000 ± 200
4700 ± 900
7000 ± 200
7800 ± 700
aCellular uptake was calculated as metal content measured in cells divided by total metal content in both cell samples and cell culture
media samples. DNA nt /mc was calculated as DNA nucleotide bases (µmol) divided by metal content measured in DNA sample
b
c
(µmol). RNA nt /mc was calculated as RNA nucleotide bases (µmol) divided by metal content measured in DNA sample (µmol).
dRuthenium levels in DNA and RNA samples were under the detection limit (< 2 ppb).
complex prodrug, compound 1, and cisplatin were tested in dose
response, along with the antibiotics rifampicin, tetracycline and
nalidixic acid. Optical density was used to quantify the response
in E. coli. The activity of 1 was evaluated both in the absence
of light and after light activation (described as “dark” and
“light”; irradiation results in the formation of compound 2;
Scheme 1). The half maximal inhibitory concentration (IC50)
value was compared with the minimum inhibitory
concentration (MIC),33 an important clinical standard
parameter32-35 that effectively defines the lowest concentration
to achieve a complete inhibition effect. As shown in Table 1,
IC50 values of 2.6 and 2.0 µM for light-activated 1 and cisplatin
were obtained in E. coli, with MIC values that were 2–3-fold
higher.
The biological activity of cisplatin and compound 1 was also
studied in human promyelocytic leukemia HL60 cells. This
relatively fast growing suspension cell line and was chosen over
adherent cell lines to more closely resemble bacterial growth
conditions. Upon light irradiation, 1 exhibited an IC50 of 3.4 µM,
similar to the IC50 of 2.6 µM for cisplatin. No cytotoxic effect
was seen for compound 1 at 300 µM in the dark, resulting in a
phototoxicity index (PI) of > 88. As expected, the cytotoxicity
of cisplatin was not affected by treatment with light. These
experiments demonstrated that light irradiated 1, like cisplatin,
is cytotoxic in both prokaryotic and eukaryotic cells, and with
very similar potencies, suggesting the mechanism of action is
through general cellular targets or biological processes present
in both cell types.
a total of 10% of the dosed compound localized in E. coli cells.
Only 6% of the dosed cisplatin was found in the cells.
Genomic DNA and total RNA isolation was performed after
24 hours of treatment, followed by AAS analysis for ruthenium
or platinum. While no ruthenium was found with either of the
nucleic acids for compound 1 in the dark, 1.3% of the ruthenium
was found with the DNA when the compound had been exposed
to light. This corresponds to a ratio of 2000 nucleotide bases per
metal center (nt / mc). Only 0.5% of ruthenium was found with
the RNA, providing a ratio of 3800 nt / mc. As a result, the
active compound 2 appears to be slightly more reactive with
DNA than RNA, with about a 1.5–2-fold difference between the
metal levels in the two nucleic acids. A similar trend of
increased reactivity with DNA over RNA was observed for
cisplatin, with 3000 nc / mc in DNA and 4700 nt / mc in RNA,
to give a 1.6-fold difference in apparent reactivity.
The uptake of the compounds was also assessed in mammalian
cells. After 24 hour treatment with 20 µM compound 1, 0.64%
of dosed ruthenium was found in HL60 cells with light
irradiation, in contrast to only 0.11% present when the cells
were kept in the dark. These results indicate that the prodrug
form is taken up much less effectively than the active species.
The metal content of the active compound in cells is comparable
to the 0.72% of cisplatin that accumulated under the same
conditions. Isolation of DNA and RNA and metal content
analysis revealed that no nucleic acid-bound ruthenium was
observed for 1 in the dark, but treatment of 1 and irradiation
resulted in 4800 nt / mc in DNA, and 5000 nt / mc in RNA. This
corresponds to 1.3% of the cellular ruthenium found with the
DNA and 2.0% in the RNA. Quantification of the metal binding
of cisplatin gave 7000 nt / mc in DNA and 7800 nt / mc in RNA
(1.1 and 1.5%). The nucleotide base to metal center ratios were
Cellular Uptake and Nucleic Acid Metallation.
Cellular uptake of the metals in E. coli was measured by atomic
absorption spectroscopy (AAS; Table 2). Light irradiation of
compound 1 resulted in a 5-fold increase in cellular uptake, with
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Figure 1. Complex 1 induces filamentous growth and decreased protein production in E. coli. Bright field and fluorescent imaging of E. coli
cells. A) N. C. control, B) cisplatin, C) compound 1 with light, D) compound 1 in the dark. Size distribution histograms of E. coli cells
associated with the conditions for A) - D): E) N. C. control, F) cisplatin, G) compound 1 with light, H) compound 1 in the dark. Histograms
of average fluorescence intensity correlated to cell size with the different treatments: I) N. C. control, J) cisplatin, K) compound 1 with light,
L) compound 1 in the dark. Cells were treated with 100 µM of each compound for 6 hours before imaging.
It has been reported by DeRose et al. that platinum accumulates
more in the cellular RNA than DNA.36 This is partly due to the
higher abundance of RNA in the cell (10–50-fold). Despite this
difference in abundance for the different nucleic acids, DeRose
demonstrated that there is a 3.8-fold preference for cisplatin to
react with DNA vs. RNA in S. cerevisiea, with 1661 nt / mc in
DNA and 6369 nt /mc in RNA after 12 hours of treatment at
100 µM.36 While our study used 20 µM of cisplatin treatment
for 24 hours, we observed the same preferential metal binding
with DNA over RNA in both E. coli and mammalian cancer
cells, though we observed a closer nt / mc ratio between DNA
and RNA. This similar binding trend across different cell types
reveals once more that cisplatin exhibits a general DNA
damaging ability in both eukaryotic and prokaryotic systems.
The similar biological accumulation characteristics of
compound 1 and cisplatin in bacterial, yeast, and mammalian
cells suggest a mechanism of action through common biological
targets or processes present in both prokaryotic and eukaryotic
cell types. In combination with extensive in vitro DNA
damaging assays,31, 37 this supports a DNA-based mechanism of
action, though multiple subsequent events may be involved that
induce the cytotoxic effects.
Phenotypic Analysis of E. coli Following Compound
Treatment.
1) Filament Size. For imaging studies, the MIC was used in
order to more closely mimic physiological treatment conditions;
data was also taken at 10x MIC. The cytological characteristics
of the E. coli were assessed, and eleongated cells were observed
after treatment with cisplatin and compound 1 with irradiation
(Figure 1). Treatment with 1 in the absence of light didn't induce
E. coli filamentous growth, and the cells were characterized by
the same short rod shaped morphology as the untreated control.
To gain a more quantitative understanding of filament
formation in populations, cells in multiple views (~200 per
condition) were chosen for size analysis. Treatment at 10x MIC
with cisplatin and compound 1 with irradiation caused a shift in
population distribution, where 68 and 73% of cells were
fillamentus for cisplatin and 1, respectively. The major
population group at 100 µM 1 were cells over 40 µm long (29%),
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Figure 2. Phenotypic profiles of compounds with different mechanisms of action in E. coli cells. Fluorescent imaging: A) Top: N. C. control;
Middle: cisplatin; Bottom: compound 1 with light; B) Top: rifampicin; Middle: tetracycline; Bottom: nalidixic acid. The merge is the
combination of the Hoechst and FM4-64 membrane stain. Colony forming experiment with various compounds: C) Left: N. C. control;
Middle: cisplatin; Right: compound 1 with light; D) Left: nalidixici acid; Middle: rifampicin; Right: tetracycline. Cells were treated with
each compound at MIC for 6 hours before imaging or colony forming. E) Quantitative and qualitative analysis of E. coli filamentous growth
and nucleoid morphology phenotypes in response to compounds treatment.
inhibits translation via binding to the 30S subunit of the
ribosome, preventing entrance of aminoacyl-tRNAs to the A-
site. These compounds were selected as agents that do not
induce DNA damage.41-43 Nalidixic acid, which inhibits
gyrase and induces DNA double strand breaks, was
investigated as a DNA damaging agent with a distinct
mechanism of action from cisplatin.44-45
All antibiotics were able to induce E. coli filaments, but the
populational size analysis revealed the major populations of
E. coli varried significantly in length. Tetracycline treatment
at the MIC resulted in a large fraction (88%) of the cell
population of normal length, with only 22% forming short (5–
10 µm) filaments. In marked contrast, nalidixic acid induced
very long filamentation, and the filaments were the only
population (100%; average length of 51 µm). For rifampicin,
a concentration of 10x MIC was required to induce any
filaments. Treatment at this concentration resulted in 30% of
the population forming short filaments of 5–10 µm; 70% of
cells were normal length (≤5 µm; Figure S2). This initial
analysis made clear that compound 1, cisplatin, and nalidixic
and only 5% of the cells were in the ≤5 µm size range. The
same trend was seen after cisplatin treatment, and the
histograms for the population of filaments are remarkably
similar (Figure 1F and G). In contrast, at the MIC, both the
filament length and % filamentous population were lower,
with only 30% and 41% of cells forming filaments for
cisplatin and 1 (Figure 2E and S4). As shown in the
histograms in Figure 1E and H, both the no treatment control
and dark control for compound 1 exhibted a dominant
population (over 97%) of cells in the ≤5 µm size range, which
represents the normal E. coli cell size. Thus, filamentation is
only associated with the light-activated form of 1.
It has been observed that many compounds induce
filamentous growth of E. coli. In order to determine if this
morphological feature corresponds to the compounds’
mechanisms of action, we compared the metal-based
compounds, preliminarily classified as DNA crosslinkers, to
two commonly used antibiotics that inhibit transcription or
translation, and one gyrase inhibitor. Rifampacin prevents
transcription by binding and inhibiting the bacterial DNA-
dependent RNA polymerase (RNAP)38-40 and tetracycline
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Figure 3. Dendra2 expression in E. coli cells. A) Dendra2 distribution in cells and fillaments; Red Dendra2 (left), Green Dendra2 (middle),
merge (right). From top to bottom: N. C. control; cisplatin (100 µM); compound 1, light (100 µM); rifampicin (3 µM); tetracycline (48 µM).
Cells were treated for 6 hours before imaging. The scale bar is 20 µM. B) Dendra2 production inhibition after 16 hours treatment. Compound
treatment only affects the production of new, green Dendra2. C) Dose response of Dendra2 production inhibition measured at 0 and 16 hours
after treatment with cisplatin (blue), compound 1 with light (red), rifampicin (green), tetracycline (black).
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acid all induced longer filaments that were a larger portion of
the population under all treatment conditions than antibiotics
that inhibited transcription or translation.
nucleoids within the cells were quite varied. In order to quantify
this observation, the % STD (the ratio of the standard deviation
to the average nucleoid size, used as a measurement of
variability; Figure S12) was calculated. The % STD was 38%
for the no treatment control, and was 36–49% for the
transcription and translation inhibitors. In the metal complex
and nalidixic acid treated systems, however, the % STD was
121-160%. Values greater than 100% indicate the standard
deviation of nucleoid size exceeded the average size of the
nucleoids. This large range of nucleoid size implicates issues of
DNA fragmentation and failure of DNA segregation after DNA
replication.46 The morphological changes in the bacterial
nucleoids treated with the metal compounds and nalidixic acid
demonstrate a multifaceted process as a consequence of DNA
damage, in contrast with compounds that act to inhibit
transcription or translation, which did not result in DNA
fragmentation.
It is well established that the processes of transcription and
translation are closely coordinated in E. coli, and the
“transertion model” posits, in part, that coupled transcription-
translation and membrane association of the growing protein
impacts nucleoid morphology. Thus, any process that interferes
with mRNA production and protein synthesis could be reflected
in the nucleoids. A recent report demonstrated that transcription
and translation inhibitors affected E. coli nucleoid shape and
spatial distribution, with expansion observed with treatment of
rifampicin and compaction with tetracycline.46 This is
qualitatively similar to our results. In addition, treatment with
nalidixic acid resulted in the observation of fragmented
nucleoids,46 similar to our imaging results with this compound
and the metal complexes. This supports our hypothesis that
nucleoid morphology can be used as a phenotypic indicator of
DNA damage.
2) Membrane Integrity. The membrane stain FM4-64 was
used to confirm that the observed filaments were single cells
and to visualize membrane integrity. As shown in Figure 2A and
B, filaments were formed by single cells upon compound
treatment. No disruption of the cell membrane was observed,
indicating that the phenotypic changes were not associated with
cell lysis. This is consistent with results that were obtained
utilizing Trypan blue staining of HL60 cells, which indicated
that neither of the two metal compounds act as membrane
damaging agents. Thus, the abnormal features observed occur
in live cells and are not an artifact resulting from physical
disruptions of cellular integrity. In addition, the mechanism of
action does not entail membrane damage.
3) Nucleoid Morphology and Number. As E. coli contain a
single chromosome, DNA staining and analysis allows for
detection of DNA fragentation or other morphological changes
due to compound treatment. Over 30 cells per treatment
condition were analyzed, and distinct effects were observed for
the impact of the different compounds on E. coli nucleoids
(Figure 2A and B).
Both rifampicin and tetracycline treatment produced filaments
with a regular distribution of DNA. Rifampicin treatment (at
10x MIC) produced the fewest nucleoids, with the majority of
filaments containing a single nucleoid that spread along the
length of the cell. Tetracycline, in contrast, produced a number
of nucleoids in each of the filaments, and the nucleoids were
compact and regularly distributed throughout the cell.
In marked contrast, nalidixic acid, cisplatin, and light
irradiated 1 caused expansion, fragmentation, and irregular
distribution of nucleoids. Both the size and distribution of the
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A colony forming assay was performed to provide further Figure 3. The transcription inhibitor rifampicin and translation
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support for the assignment of a DNA damaging mechanism of
action (Figure 2C and D). Cells were treated at the MIC for each
compound, and then media removed and the cells were spread
on an agarose plate. Only cells treated with the transcription
and translation inhibitors were able to form colonies; the metal
complexes and nalidixic acid were clearly cytotoxic at their
MIC. This supports a conclusion that these three compounds
induce irreversible damage to the E. coli, likely through DNA.
4) Protein Production. Cisplatin and other platinum-based
agents are known to interfere with protein production. Some
question remains, however, if this is an important feature that
induces cell death, or simply a side effect of the DNA damage.
Several experiments have quantified the impact on protein
production after transfection of already metalated plasmids into
living systems.47-48 To study the process and impact of DNA
metalation, we treated E. coli with the metal complexes and
subequently monitored protein production. This experiment
couples the quantitation of protein levels in the detection of the
fluorescent protein to the preceeding natural sequence of events
that imapact transcription/translation, and allows for
observation of important features that may play a role, such as
compound uptake, localization within the cell, or sequence-
dependant interactions with the nuclic acid.
Cells undergoing death will slow or cease protein production,
which produces a similar phenotype to cells that are under the
influcence of a transcription or translation inhibitor. To
discriminate between inhibition of protein production and
induction of cell death, we used a photo-convertible protein,
Dendra2, as a reporter, since it is able to provide information on
both aspects of cell viability and new protein production
simultaneously.49 Dendra2 undergos a photochemical reaction,
transforming from a green fluorescent protein to a red
fluorescent protein when exposed to 405 nm light. The
photoconverted "Red" Dendra2 emission provided a stable
internal reference for cell health and cell number for all samples,
while new protein production (after light exposure) is reflected
in the "Green" Dendra2 emission. Both forms are stable and
persist in living cells with half-lives (t1/2) on the order of 50 to
70 hrs.50-52 The two forms of the protein thus provide spatial and
temporal tracking of Dendra2 formed before and after light
exposure.
Dendra2 production was induced in E. coli with IPTG and
allowed to proceed for 3 hours before photoconversion,
followed by compound treatment. A clear negative correlation
was seen between protein production and cell size, where
filamentous cells with longer fliament lengths exhibited a lower
fluorescence intensity, reflecting a reduction in the amount of
new Dendra2 protein being produced. As shown in Figure 1,
after 24 hours of treatment with compound 1, the average
fluorescence intensity of the cell population with the largest
length (> 40 µm) dropped by over 70% compared to the control
population. Other populations with increased cell lengths
exhibited a 30 – 70% decrease in fluorescence intensity. The
same trend was seen in cisplatin treated cells, where the
fluorescence intensity decreased by 15 – 80%, depending on the
length of the filament. Both compound dose and the time of
treatment was found to have an effect on filament formation and
protein production (see Figure S3 - S5).
inhibitor tetracycline exhibited IC50 values for inhibition of
Dendra2 production that matched well with growth inhibition
(within 3 – 10-fold; see Table 1 and Figure 3). In contrast, both
compound 1 and cisplatin displayed a greater disparity between
inhibition of protein production and cell growth inhibition. The
30 – 40-fold decrease in potency reflects that the mechanism of
action of cisplatin and compound 1 is not solely (or primarily)
through transcription or translation inhibition. In contrast,
nalidixic acid, which induces DNA double strand breaks, was
far more effective at inhibiting protein production.
While cisplatin has been described as a transcription inhibitor,
it was the least effective of the five compounds tested for
inhibition of protein production. The impact of the DNA
damage induced by platinum compounds on protein production
has been comprehensively and conclusively proven, along with
the restoration of protein production when the appropriate DNA
repair mechanisms are activated to remove the lesions. However,
our studies suggest that the inhibition of protein production by
cisplatin is of secondary importance for the health of E. coli, as
the concentrations required to observe a significant impact on
this process far exceeded the toxic dose for the compound.
In an analogous study, Lippard and coworkers tested cisplatin
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in mammalian cells containing
a genetically encoded
fluorsecent reporter system.53 Very good agreement was
observed between the concentrations required to inhibit protein
syntheis and to induce cytotoxicity evaluated via a colony
counting assay in that report. The reason for the disparity in the
ability of cisplatin to inhibit protein production in E. coli
compared to the HeLa cells used by Lippard is unclear. It is
particularly surprising, given the very similar values we found
for DNA and RNA metallation in E. coli and HL60 cells, as
described above. However, due to the sensitivity of DNA
damage detection mechanisms in mammalian cells, it seems
unlikely that translation would be affected before initiation of
the DNA damage response.
5) Protein distribution. The use of a photoconvertable protein
allows for a spatiotemporal analysis of protein content. This
provided the opportunity to address intriguing questions such as
the impact of interruption of cell division and filamentous
growth on the activity of ribosomes for new protein production,
and the re-distribution of existing protein within a filamentous
cell. Fluorescent imaging was performed to probe the effects of
the different compounds on protein distribution in single cells.
Compounds were dosed after Dendra2 photoconversion, and
imaging was performed 6 hours later. As shown in Figure 3 A,
both the "Red" Dendra2 (the internal control of pre-treatment
protein level) and the "Green" Dendra2 (reflecting protein
synthesis after treatment) were distributed throughout the cell
as the healthy cells underwent multiple cell divisions. Both Red
and Green forms of Dendra2 were also found within the
filamentous cells where cell division was blocked by either
DNA damaging agents or transcription/translation inhibitors. It
has been reported that disruption of DNA replication and double
strand breaks resulting from nalidixic acid treatment could lead
to uneven distribution of ribosomes in filamentous cells.46
However, we did not observe any particular spatial
sequestration of active ribosomes; alternatively, protein
diffusion is sufficiently rapid to prevent observation of any
localization during the time scale of the experiment.
The production of Dendra2 was quantified by dose response,
providing IC50 values for inhibition of protein production.
Protein production was quantified using the ratio of the average
fluorescent intensity of the two forms of Dendra2, as shown in
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Figure 4. Compound 1 induces apoptosis in HL60 cells without cell cycle arrest. A) Flow cytometry by PI/Annexin V in HL60 cells; Red =
apoptotic cells, Blue = necrotic cells, Black = dead cells. B) Flow cytometry by PI in HL60 cells; Black = G1; Red = G2; White = S phase.
C) Immunoblotting of cleaved PARP and cleaved caspase 3 in HL60 cells. GAPDH was blotted as loading control. D) Agarose gel
electrophoresis of DNA laddering. HL60 cells were treated for 24 hours for flow cytometry. All pannels: compound 1, 20 µM; cisplatin, 20
µM; doxorubicin, 1 µM.
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This analysis of the ratio of DNA or RNA bases to metal
Comparison of In Vitro and In Cell Protein Production.
centers suggests the functional inhibition of protein synthesis
Previously, we reported an in vitro transcription and
by covalent adducts to DNA and mRNA by compound 1 and
translation assay (IVTT) with compound 1 and cisplatin using
cisplatin is similar in E. coli and the in vitro assay. It is notable
Green Fluorescent Protein (GFP) as a fluorescent reporter.37 In
that in a living cell, where the reaction conditions are much
this assay, either a plasmid containing the GFP gene or the
more complex than the buffered system of IVTT assay, the IC50
mRNA transcript for GFP were allowed to react with varying
concentration of cisplatin or compound 1, with irradiation,
values to inhibit protein synthesis were diminished by over 60-
fold relative to the IVTT assay. However, the ratio between
before addition of the nucleic acids to a cancer cell lysate
DNA or RNA bases and the metal center for inhibition of
containing transcription and translation machinery. Both metal
protein production remain quite consistent. The increase in the
compounds inhibited GFP production with a clear dose
IC50 values in cells suggests two conclusions: 1) inhibition of
response. Interestingly, the IC50 value for inhibition of protein
production was ~ 3 µM for both compounds. The ratio of DNA
nucleotides or RNA nucleotides to each metal center was
protein synthesis is not the factor that induces cell death, and 2)
both compounds suffer from off-target binding to biological
molecules. The later is known to be a major issue for many
calculated at the IC50 for protein synthesis inhibition. The values
currently administered drugs, especially cisplatin.
for compound 1 were 1140:1 and 820:1, while the values for
cisplatin were 600:1 for DNA and 610:1 for RNA respectively.
In the current uptake studies, E. coli cells were dosed at 20
The role of off-target binding was also supported by the AAS
analysis of metal content with the different nucleic acids in E.
coli and mammalian cells, as only a minor component of the
µM, which is approximately 10x higher than the IC50 value for
metal compounds entered the cells, and of this, only 1.30% and
growth inhibition, but well below the IC50 value for inhibition
of protein synthesis, as determined by Dendra2 production (see
Table 1). In order to compare the in vitro experiment to the cell
data, extrapolation of the ratio for DNA and RNA nucleotides
per metal center at the IC50 value for in vivo protein synthesis
inhibition was performed as detailed in the Supporting
Information. The calculated values were remarkably close to
the values from the in vitro transcription and translation assay,
1.26% of cellular ruthenium from compound 1 and 0.98% and
1.12% of the cellular platinum from cisplatin were found with
genomic DNA in the two systems. If one includes the < 2% of
metal present in the RNA as on-target damage, this means over
96% of the cellular metal is reacting with potentially non-
relevant targets. Extending this argument, if off-target binding
could be eliminated, cytotoxicity IC50 values would be reduced
to nanomolar concentrations if the same levels of cellular
with DNA nucleotides to metal center ratios of 520:1 for
uptake could be maintained. This may lead to another method
compound 1 and 700:1 for cisplatin. The RNA nucleotide to
metal center ratio was 1000:1 for compound 1 and 1090:1 for
cisplatin.
to improve the potency of currently used drugs: instead of
focusing on the generation of analogues that are more potent
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against nucleic acids, analogues with reduced off-target binding cisplatin and doxorubicin were able to induce apoptosis in the
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could be more effective.
absence of functional p53 in HL60 cells (Figure 4), the A549
cell line demonstrated clear induction of p53 for these two
compounds. In contrast, compound 1 did not induce elevated
expression of p53, and didn't significantly alter its
phosphorylation or expression of p21 (Figure 5A).
Another surprising difference observed between the platinum
and ruthenium compounds is that p-chk1, which is involved in
G2/M cell cycle arrest in response to DNA damage, was not
induced by treatment of compound 1 in either cell line, while
both cisplatin and doxorubicin induced phosphorylation of chk1.
This finding is consistent with the fact that no cell cycle arrest
point was seen with compound 1 in HL60 cells, in contrast to
cisplatin and doxorubicin. However, phosphorylation of γ-
H2AX, an early sensor of DNA damage, was observed after 6
hours of treatment with 1, indicating DNA damage even in the
absence of chk1 activation.
It has been shown that the vast majority of cisplatin and other
Pt species bind to plasma proteins and do not reach their target
in vivo.54-56 It was anticipated that the ruthenium compound
would fare better than cisplatin in avoiding off-target binding,
due to its lower affinity for hydrophobic proteins such as human
serum albumin (HSA)37 and thiols such as glutathione (GSH),
but this has not been found to be the case in cells.31, 57 As the
preferred binding partners are not the same for the platinum and
ruthenium complexes, it will be important to identify the
primary off-target biomolecules responsible for sequestering
the ruthenium in order to rationally design derivatives that avoid
these species to increase the potency of these inorganic
compounds.
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Ru and Pt Compounds Induce Distinct Cellular Re-
sponses in Mammalian Cells.
Pro-survival and pro-apoptotic pathways, including MAPK
pathways were examined, and consistent signaling behaviors
after compound 1 treatment were observed in both cell lines.
The ERK pathway has been reported to facilitate cell survival
and prevent apoptosis.62-63 As shown in Figure 5, this pathway
was inactivated by compound 1 in both A549 and HL60 cell
lines; in contrast, both cisplatin and doxorubicin didn't down-
regulate this pro-survival pathway. The JNK pathway has been
reported to act as a pro-apoptotic pathway in response to
cellular stress induced by DNA damage, and is mainly activated
by mismatch repair signals.62, 64 Both cisplatin and doxorubicin
were able to induce phosphorylation of JNK in A549 and HL60
cells at 24 hours, though different phosphorylation levels in
HL60 cells were observed, which might indicate possible
phosphorylation time course differences. Phospho-JNK was
seen as early as 6 hours after doxorubicin treatment in HL60
cells while cisplatin induced phospho-JNK was seen to increase
to maximum level at 24 hours.65-66 Surprisingly, compound 1
didn't activate the JNK pathway to the same extent as cisplatin
or doxorubicin. The phosphorylation level was slightly
increased within 6 to 12 hours of treatment with compound 1 in
both cell lines, but then decreased over time. This, along with
the previously discussed markers, indicate a different DNA
damage response for compound 1 either from altered cell
signaling pathways or by a different class of DNA damage.
The cellular effect of compound 1 was also studied in
mammalian cells, with a focus on proteins involved in cell
signaling and cell death. As shown in Figure 4, effects on cell
cycle and apoptosis were studied using flow cytometry,
immunoblotting for apoptotic markers, and DNA fragmentation.
No cell-cycle specific arrest point was observed with compound
1 treatment, while a sub G1 population of 20% of cells was
observed after 24 hours. Flow cytometry analysis of apoptosis
vs. necrosis using FITC-AnnexinV and propidium iodide
indicated that compound 1 induced cell death through apoptosis
as the dominant mechanism. While cisplatin induced necrosis
in a small fraction (5%), less than 2% of cells treated with
compound 1 were characterized as necrotic. Immunoblotting of
caspase 3 and PARP showed a time dependent induction of
apoptosis; in addition, isolation of genomic DNA showed
fragmentation, which is consistent with apoptotic cell death. All
apoptotic reporters were clearly observed at 24 hours.
The tumor suppressor protein p53 regulates cell growth and
cell cycle checkpoints to eliminate proliferation. It is one of the
most commonly mutated genes in cancer, resulting in loss of its
regulatory function.58-59 Both p53/p21 and chk1 are involved in
G1/S and G2/M cell cycle checkpoints in response to DNA
damage.60-61 In order to probe the role of p53 in response to
compound 1, immunoblotting was performed in A549 cells.
This non-small-cell-lung cancer cell contains functional p53, in
contrast with the p53 deficient HL60 cell line. While both
Figure 5. Immunoblotting of apoptotic markers and cell signaling proteins in A) and B) A549 cells; C) HL60 cells. Cells were treated with
20 µM of compound 1 for specified time periods, cisplatin (20 µM) and doxorubicin (2 µM) at 24 hours of treatment were used as controls.
GAPDH was used as loading control.
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recognition and repair pathways that are unique to eukaryotic
Recent reports have raised questions as to the source of the
systems are implicated as playing a key role in their mechanism
of action? 2) While more of the metal complexes are taken up
in E. coli than the HL60 cells, the nt / mc ratio remains quite
consistent for both DNA and RNA. What biological entities are
responsible for the enhanced sequestration of the metals in the
E. coli, and is it possible that similar molecules play a role in
cisplatin-resistant cancer cells? 3) Why does DNA packing not
play a greater role in determining the degree of metallation?
DNA is packaged in different ways and to different degrees of
compaction in the two cells types, and if the more highly
exposed, transcriptionally active seuqences were the primary
target, we would anticipate greater potency for inhibition of
Dendra2 production. We believe that adressing these basic
questions may be very important to the rational development
of improved DNA targeting agents, and see E. coli as a
excellent system to seek the answers.
biological effects of compound 1 and analagous Ru(II) systems,
with data suggesting that it is the liberated bipyridyl ligand,
rather than the Ru(II) metal center 2, that is responsible for
activity.67-68 This study demonstrates such striking similarities
between the behavior of the light activated Ru(II) complex and
cisplatin, both in E. coli and mammalian cancer cells, that we
find it improbable that the ligand, rather than the metal center,
is responsible for the phenotypic effects. The cytological
profile, nucleic acid metallation, and DNA damage response is
consistent with metal-mediated DNA damage. However, it is
possible that the ligand is inducing other effects that are not
observed with these assays. A more detailed investigation is
underway.
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DISCUSSION
This work demonstrates that a combination of phenotypic
screening based on E. coli imaging and protein production
using Dendra2 as a fluorescent reporter allows for rapid
investigations of mechanisms of action for cytotoxic agents
which may have similar activities in mammalian cells. We
found that a combination of these two experimental parameters
facilitates discriminating DNA damaging agents from agents
that work solely as transcription or translation inhibitors. While
filaments are formed by all classes of compound, filament size
and population distribution was radically different depending
on the mechanism of action. Furthermore, the observation of
irregular bacterial nucleoids, easily visualized using Hoechst
staining, was associated with DNA damage, while regular
nucleoid size, shape, and distribution was found with
compounds that do not directly affect DNA. Such experiments
could not performed in eukaryotic cells, given the number of
chromosomes.
E. coli have been used in the past as model systems to probe
binding characteristics of drugs, such as cisplatin, with proteins
using NMR.69-70 Phenotypic analysis in bacteria by microscopy
is now gaining more attention, primarily to identify the cellular
pathways impacted by antibiotics,71-72 but a recent report
identified anticancer activity for a molecule characterized by
cytological profiling in bacteria.73 In that case, identification of
the mechanism of action of the molecule under investigation
motivated later studies in cancer cells, and the authors
characterized bacterial cytological profiling (BCP) as a tool for
drug repurposing.
ASSOCIATED CONTENT
Supporting Information. Supporting figures and tables. This ma-
terial is available free of charge via the Internet at
http://pubs.acs.org.”
AUTHOR INFORMATION
Corresponding Author
* ec.glazer@uky.edu
Author Contributions
The manuscript was written through contributions of all authors. /
All authors have given approval to the final version of the manu-
script. / ‡These authors contributed equally.
ACKNOWLEDGMENT
This work is supported by the National Institutes of Health
(R01GM107586). YS was the recipient of a University of Ken-
tucky Opportunity Fellowship. All metal uptake studies are done
with technical support of Environmental Research Training Labor-
atories (ERTL) at the University of Kentucky. Experiments were
performed at the UK Flow Cytometry & Cell Sorting core facility,
which is supported in part by the Office of the Vice President for
Research, the Markey Cancer Center and an NCI Center Core Sup-
port Grant (P30 CA177558) to the University of Kentucky Markey
Cancer Center.
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