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Disruption of microtubule function in cultured human cells by a cytotoxic ruthenium(ii) polypyridyl complex.
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Disruption of microtubule function in cultured
human cells by a cytotoxic ruthenium(II) polypyridyl
complex†
Nagham Alatrash,a Faiza H. Issa,a Nada S. Bawazir,b Savannah J. West,c Kathleen E. Van
Manen-Brush,b Charles P. Shelor,a Adam S. Dayoub,a Kenneth A. Myers,b
Christopher Janetopoulos,b Edwin A. Lewisc and Frederick M. MacDonnell *a
Treatment of malignant and non-malignant cultured human cell lines with a cytotoxic IC50 dose of 2 mM
tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II) chloride (RPC2) retards or arrests microtubule motion
as tracked by visualizing fluorescently-tagged microtubule plus end-tracking proteins.
Immunofluorescent microscopic images of the microtubules in fixed cells show substantial changes to
cellular microtubule network and to overall cell morphology upon treatment with RPC2. Flow cytometry
with MCF7 and H358 cells reveals only minor elevations of the number of cells in G2/M phase,
suggesting that the observed cytotoxicity is not tied to mitotic arrest. In vitro studies with purified tubulin
reveal that RPC2 acts to promote tubulin polymerization and when imaged by electron microscopy,
these microtubules look normal in appearance. Isothermal titration calorimetry measurements show an
associative binding constant of 4.8 106 M1 for RPC2 to preformed microtubules and support a 1 : 1
RPC2 to tubulin dimer stoichiometry. Competition experiments show RPC2 does not compete for the
Received 8th November 2019
Accepted 11th November 2019
taxane binding site. Consistent with this tight binding, over 80% of the ruthenium in treated cells is coDOI: 10.1039/c9sc05671h
localized with the cytoskeletal proteins. These data support RPC2 acting as an in vivo microtubule
rsc.li/chemical-science
stabilizing agent and sharing many similarities with cells treated with paclitaxel.
Introduction
Microtubules (MTs) play an essential role in mitosis, cellular
structure, and trafficking, as well as offer a promising target for
innovative chemotherapeutic agents.1,2 MTs are composed of
ab-tubulin heterodimers that undergo highly regulated and
dynamic bouts of polymerization and depolymerization.
Microtubule targeting drugs disrupt this ‘dynamic instability’
by inhibiting or promoting polymerization and interfere with
mitosis and other essential cellular processes, leading to
apoptosis.3,4 Agents that inhibit tubulin polymerization, such as
nocodazole (NCZ), vincristine, and colchicine, are known as
microtubule destabilizing agents (MDAs) and are used therapeutically.5,6 Microtubule stabilizing agents (MSAs), more
recently discovered, inhibit MT depolymerization. The rst
identied MSA, paclitaxel (PTX, Taxol), was discovered in 1979,7
and approved for clinical use in 1993 for treatment of solid
a
Department of Chemistry and Biochemistry, University of Texas at Arlington,
Arlington, TX, 76019, USA. E-mail: macdonn@uta.edu
b
Department of Biological Sciences, University of the Sciences, Philadelphia, PA 19104,
USA
c
Department of Chemistry, Mississippi State University, Starkville, MS 39762, USA
† Electronic supplementary
10.1039/c9sc05671h
information
264 | Chem. Sci., 2020, 11, 264–275
(ESI)
available.
See
DOI:
tumor malignancies.4,8–10 Since this time a number of synthetic
derivatives, e.g. docetaxel, or new types of MSAs, such as epothilones, laulimalide, discodermolide, cetamine A and B, dictyostatin, peloruside A, dactylolide, and zampanolide, have
been discovered; most of which are natural products believed to
have evolved as broad-spectrum toxins to target MTs in prey or
predators.8,11 These MSAs are complex organic molecules with
multiple fused ring structures and chiral centers in which the
regio and stereochemistry must be controlled. Their large-scale
synthesis is oen a major undertaking and an important
consideration in their development.
The only reports of metal complexes targeting microtubules
in cells are limited to the activity of simple ions, such as As3+,
Pb2+, and Hg2+,12–14 and one organometallic compound, Os3(CO)10(NCMe)2.15 The simple ions generally destabilize MTs
whereas the trinuclear osmium carbonyl cluster is proposed to
lose the two labile MeCN and react with tubulin sulydryl
groups to induce hyperstabilization of the MT structure. Simple
and ubiquitous ions like Mg2+ and Ca2+ are required for MT
polymerization and depolymerization16–18 and at elevated,
usually non-physiologically relevant, concentrations of these
cations cause MT depolymerization.19 We and others have reported on the potent cytotoxicity of [Ru(DIP)3]Cl2 (Fig. 1) in
numerous malignant (H358, MCF7, CCL228, HL60, B16, MDAMB-231, A549, Jurkat, ML2, SF) and non-malignant (MCF10a)
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Fig. 1 Chemical structure of tris(1,10-phenanthroline)ruthenium(II)
(RPC1) and tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II) (RPC2)
cations. These ruthenium(II) polypyridyl complexes are used as the
chloride salts in this study.
cell lines.20–23 IC50's consistently range between 1 and 4 mM
irrespective of the cell type. Conversely, the structurally similar
but smaller [Ru(phen)3]Cl2 (Fig. 1) rarely shows any cytotoxicity
below 50 mM.20 Herein, we show that these two coordination
complexes promote tubulin polymerization in vitro, and, more
signicantly, RPC2 binds MTs in vivo (live cells) and induces
massive changes to the MT structure and dynamics. This MT
disrupting activity correlates with the observed cytotoxicity and
supports this as the dominant apoptotic mechanism of action.
Experimental
Materials and methods
Reagents. Ruthenium(III) chloride trihydrate (Pressure
Chemical Co) was used as received, tetrabutyl ammonium
chloride hydrate, 4,7-diphenyl-1,10-phenanthroline (DIP), 1,10phenanthroline (phen), ammonium hexauorophosphate,
ethanol, acetonitrile (Aldrich) were used as received. [Ru(1,10phenanthroline)3]2+ (RPC1) was synthesized according to literature.24 PTX, tubulin porcine brain (>99% pure), general tubulin
buffer and glycerol tubulin buffer were purchased from Cytoskeleton Inc., (Denver. CO). NCZ was purchased from Sigma
Aldrich. The complete EDTA-free Protease Inhibitor Cocktail
Tablets was purchased from Roche.
Synthesis of [Ru(DIP)3]Cl2 (RPC2). This complex was
prepared by using a modied literature procedure.24,25 DIP
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(5.6 g, 17 mmol) and RuCl3$xH2O (38% Ru by mass) (0.74 g, 2.8
mmol) were suspended in 150 mL of ethanol. Aer reuxing
overnight, the mixture was cooled to room temperature, ltered,
and the volume reduced to 50 mL by rotary evaporation. The
product was precipitated by adding an aqueous ammonium
hexauorophosphate (NH4PF6) dropwise until a precipitate was
clearly present. The precipitate was ltered and washed with
ethanol followed by washing with copious amount of water and
dried in vacuo at 60 C for 12 h. Yield 3.0 g of the hexauorophosphate salt (83%). The NMR data are identical to that
reported in the literature 1H NMR (CD3CN) d ¼ 7.59–7.62 (m,
30H, HDIP), 7.64 (d, 6H, JHH ¼ 5.7 Hz, H3, H8), 8.20 (s, 6H, H5,
H6), 8.25 (d, 6H, JHH ¼ 5.1 Hz, H2, H9).
The chloride salt was prepared from the hexauorophosphate salt by dropwise addition of a concentrated
solution of tetra-n-butylammonium chloride in acetone to
a concentrated solution of the [Ru(DIP)3][PF6]2 salt in acetone.
The resulting precipitate was ltered and washed with acetone,
then dried in vacuo at 60 C. Yield 2.25 g (95%) [Ru(DIP)3]Cl2.
Preparing RPC2 aqueous solutions. RPC2 (chloride salt) is
sparing soluble in water or buffer unless it is rst dissolved in
a small amount of DMSO and then diluted into the aqueous
solution. All RPC2 solutions in this work were prepared in this
manner and contain 5% or less DMSO by volume. Vehicle only
solutions contain the same amount of DMSO as the RPC2
solutions. When prepared this way, RPC2 solutions readily
passed through a nylon lter (0.2 mM pores), and showed no
light scattering (Tyndall effect). In order to avoid precipitation,
freshly prepared solutions of RPC2 were used in all cases.
Tubulin polymerization assay. Tubulin polymerization was
determined according to the manufacture's protocol by
a biochemical Optical Density-based assay (Cytoskeleton, Inc.,
CO). Briey, tubulin porcine brain (>99% pure Cat# T240-DX)
was reconstituted with ice-cold buffer (80 mM PIPES pH 6.9,
2.0 mM MgCl2, 0.5 mM EGTA, with 10 mL of 100 mM GTP and
kept on ice). Tubulin stock solutions (10 mg mL1) were aliquoted into 5 200 labeled tubes and immediately drop-frozen
in liquid nitrogen and stored at 70 C. Polymerization was
started by adding 100 mL volume of 3 mg mL1 tubulin in
80 mM PIPES pH 6.9, 0.5 mM EGTA, 2 mM MgCl2, 1 mM GTP,
10% glycerol buffer to pre-warmed 96 well half area plate at
37 C, and followed by absorbance readings at 340 nm over 60
minutes at 37 C (temperature-controlled microtiter plate
reader). Absorbance at 340 nm was determined using FLUO-star
Omega microplate reader (BMG Labtech) in kinetic absorbance
mode. Polymerization reactions included control (control
minus ligand or anti-cancer agent vehicle), and 10 mM of PTX,
NCZ, RPC1 and RPC2. OD is proportional to the concentration
to the polymerized tubulin. The experiment was performed in
triplicate (mean values are presented). Data were exported and
polymerization plotted using Excel soware. At concentrations
of 1 mM or greater, RPC2 has an appreciable absorbance at
340 nm, therefore this was measured and subtracted to
normalize the data.
Electron microscopy. Aliquots of polymerized tubulin were
placed on 300-mesh carbon-coated, Formvar-treated copper grids
and immediately stained with 5–10 successively applied drops of
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1% (w/v) uranyl acetate. Excess stain was wicked from the grids
with torn lter paper. The grids were examined in a Hitachi H9500 High-resolution Transmission Electron Microscope.
Immunoblotting. Cells are treated with drugs for 4 h in 37 C
incubator. The conuent 60 mm dishes of cells were washed 3
times with 37 C PBS, then lysed in microtubule stabilization
buffer (Tris–HCl pH 6.8, 20 mM, NaCl 140 mM, MgCl2 1 mM,
NP40 0.5%, EGTA 2 mM, 10 mM of Taxol) and protease inhibitors (100 Protease Inhibitor Cocktail (PIC) and 100 phenylmethane sulfonyl uoride (PMSF)). Then, the cells were
scraped, mixed and quickly transfer to microtubes. These
samples were then spun at 10 000 rpm for 20 minutes, the
supernatants containing free tubulin were collected and
a bicinchoninic acid (BCA) protein assay was performed to
check the protein quantity. Equal amounts of protein sample
were prepared in Laemmli sample buffer and were boiled for
5 min at 95 C. Proteins were resolved on a 12% polyacrylamide
gel. The proteins were transferred to a nitrocellulose membrane
for 1.5 h at 100 V. The nitrocellulose membrane was stained for
1 min with Ponceau Red to verify the efficiency of the transfer.
Then, the membrane was blocked in 5% skim milk for 1 h at
room temperature and incubated with primary alpha tubulin
monoclonal antibody in 5% BSA overnight at 4 C. The
membrane was washed 3 times with TBST buffer before being
probed with secondary antibodies conjugated to HRP, then the
membrane was incubated for 1 h at room temperature before it
was washed 3 times again with TBST buffer. Protein bands were
visualized with an enhanced chemiluminescent (ECL) system.
Isothermal titration calorimetry. ITC experiments were performed using a Microcal VP-ITC (Malvern) instrument. For
polymerized porcine brain tubulin the calorimeter was set at
37 C. For depolymerized porcine brain tubulin the calorimeter
was set at 4 C. Tubulin concentration was kept below 3 mg
mL1 to ensure that minimal microtubule formation before any
polymerization assays were performed in preparation for ITC
experiments. Reverse titrations were used in all ITC experiments due to the low solubility of RPC2 in aqueous solutions. A
typical ITC experiment involved fourteen 20 mL injections of
tubulin (30 mM heterodimers, or 2 mg mL1) into a 1.45 mL
cell of ligand solution. The ITC thermograms were corrected for
titrate and titrant dilution effects by performing the appropriate
blank experiments and correcting the observed heats by subtracting the heats of dilution. Corrected ITC titrations were t
with a nonlinear regression algorithm using CHASM, an ITC
data analysis program developed in the Lewis laboratory to
determine the thermodynamic parameters, including the
association constant (K) and changes in free energy (DG),
enthalpy (DH), and entropy (TDS).
Subcellular localization assay. H358 cell line was seeded in
60 60 mm dishes and grown to 80% conuency not to exceed
5 106 cell density. The cell line was then treated with 20 mM
concentrations of RPC1 or RPC2 for 12 h. The cells were washed,
trypsinized, and centrifuged for 5 min at 1000 g to create cell
pellets. Pellets were washed 3 times with ice cold phosphate
buffer saline (PBS). The cell pellet was then fractionated using
a Qproteome Cell Compartment Kit (Qiagen, Germany)
following the procedure outlined in the associated handbook,
266 | Chem. Sci., 2020, 11, 264–275
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dated October 2012. Four protein fractions were obtained:
cytosolic proteins, membrane proteins, nuclear proteins, and
cytoskeletal proteins. The mitochondria, Golgi apparatus, and
endoplasmic reticulum are isolated in the membrane protein
fraction. Each fraction was diluted to a total volume of 5.00 mL
with a solution of 1% HNO3 water in ultrapure water. Solutions
were analyzed for ruthenium ion concentration using ICP-MS.
Indirect immunouorescence analysis. Cells were cultured
on coverslips and placed in individual wells of six-well plates.
Aer overnight incubation, cells were treated with drugs for
12 h xed with 4% paraformaldehyde. Fixed cells were permeabilized using 0.25% Triton X-100 in PBS, followed by
blocking with 5% BSA, then probed with alpha-tubulin antibody
and incubated over night at 4 C. Next day, Alexa Fluor 488conjugated goat anti-mouse IgG (H + L) secondary antibody was
administered for 2 h at room temperature followed by staining
the nucleus with propidium iodide (PI) for 5 minutes at room
temperature. The immunolabeled coverslips were mounted on
glass slides. Microscopic images were obtained using Zeiss LSM
510 with 63 oil objective. PI (lex ¼ 514 nm) and Alexa Fluor
488-conjugated (lex ¼ 488 nm) were collected at 530–617 and
505–530 nm respectively.
Cell cycle analysis. Flow cytometric analysis of cellular DNA
content was obtained using a Propidium Iodide Flow Cytometry
Kit (ab139418, Abcam). MCF7 and H358 cell line were seeded in
60 60 mm dishes and grown to 80% conuency not to exceed
6 105 cell density. The cell lines were then treated with RPC2
for 12 h at 37 C. The cells were trypsinized and centrifuged at
500 g for 5 min, washed with PBS, xed in 66% ice cold ethanol
at 4 C for 2 h. Fixed cells were centrifuged at 500 g for 5 min
and washed with PBS and resuspended in 200 mL of PBS containing 50 mg mL1 PI and 550 U mL1 RNase for 30 min at
37 C, and subjected to ow cytometry (BD LSR II ow cytometer). DNA histograms were analyzed using DIVA soware.
Spinning disk confocal imaging and image processing.
MCF7 cells were cultured in phenol-red DMEM media
(HyClone™, GE healthcare) supplemented with 10% FBS and
penicillin/streptomycin at 37 C in 5% CO2. MCF10A cells were
cultured in low-calcium phenol-red DMEM/F12 (HyClone™, GE
healthcare) supplemented with nal concentration of 5% horse
serum, 20 ng mL1 EGF, 0.5 mg mL1 hydrocortisone, 100 ng
mL1 Cholera toxin, 10 mg mL1 insulin, and 1% pen-strep. For
live imaging, 500 000 cells per mL of both MCF7 and MCF10A
cells were transfected with the cDNA (EGFP-EB3), and then
cultured on 10 mg mL1 bronectin coated 35 mm glass-bottom
dishes (Cellvis, cat#: D35-20-1.5-N). Transfection of EGFP-EB3 was
performed using a Lonza Nucleofector Device with Ingenio electroporation kit (Cat# MIR 50112, Mirus). Cells were treated with
RPC2 18–24 hours post-transfection to allow time for cDNA
expression. To minimize photobleaching, phenol-free DMEM was
supplemented with 10% FBS and 25 mM HEPES, pH ¼ 7.2
replaced the original media and was administered before RPC2
treatment. Time-lapse imaging was obtained before, 30 min, 1 h,
2 h, and 3 h aer RPC2 treatment for both MCF7 and MCF10A.
Images were obtained on a Zeiss Axiovert (Jena, Germany)
microscope with a Zeiss alpha Plan-Fluar 100, 1.45 NA oil
objective and a spinning disk confocal scan head (Yokogawa CSU-
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X1, Tokyo, Japan). A 488 nm laser source with 488/25 nm, excitation lter was used for the illumination of GFP. Digital images
were captured sequentially by a high speed EM-CCD camera
(Photometrics Evolve™ 512, Tucson, AZ). Time-lapse experiments
were automated by soware (Slidebook 6.0.9, 3i, Denver, CO).
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Results
General properties of ruthenium polypyridyl complexes
(RPCs)
While there are numerous types of ruthenium polypyridyl
complexes, one of the most commonly explored subtypes are the
tris-chelate complexes which are coordinatively saturated and
kinetically inert. RPC1 and RPC2 are two such complexes which
exhibit exceptional chemical stability and which appear to be
metabolically stable as rats and mice given intraperitoneal
injections of radiolabeled [106Ru(phen)3][ClO4]2 excrete the intact
cation in the urine in about 12 h.26 Additional examples of this
exceptional stability include surviving in boiling concentrated
acids or alkalis,27 or the use of RPC1 as a digestive marker for
ruminant animals since it is not absorbed by the intestine and
passes through the animal.27–29 This chemical robustness is one
of the reasons that RPCs are so extensively studied as biological
probes or therapeutics.30–32 In general, the complex should be
considered as robust as the free ligands excepting when irradiated which can lead to ligand loss.33,34 Importantly, the tris
chelate RPCs, such as RPC1 and RPC2, are chiral (propeller
molecules with D3 point group symmetry) and are generally isolated as a racemic mixture of L and D enantiomers. No attempt to
work with enantiopure complexes was made in this work.
RPC2 co-localizes with the cytoskeletal proteins
Ruthenium uptake by H358 cells incubated with 20 mM of RPC1
or RPC2 for 12 h at 37 C reveals 2 ng Ru per million cells for
RPC1 and 15 ng Ru per million cells for RPC2 (Fig. 2A). Additional studies were performed at 4 C and gave similar uptake,
supporting passive transport35 as the primary pathway for RPC1
and RPC2 entering cells (Fig. 2B). Passive diffusion through the
cell membrane has been shown for the closely related RPC,
[Ru(DIP)2dppz]2+ (dppz ¼ dipyridophenazine), in HeLa cells,
and appears to be a common transport mechanism for this
class of compounds.23,35–37 The ruthenium uptake for
[Ru(DIP)2dppz]2+ was reported to range between 2 to 50 ng Ru
per million cells, depending on the incubation media. This is an
excellent agreement with our data considering the cell line
differences. Assuming an average cell volume of 1.7 pL (ref. 38)
and homogenous distribution in the cell, intracellular ruthenium concentrations range from 16 to 400 mM for [Ru(DIP)2dppz]2+, 15 mM for RPC1 and 112 mM for RPC2.
In order to assess the intracellular Ru distribution, treated
H358 cells were fractionated into four components: nucleus,
cytosol, membrane proteins, and cytoskeletal proteins. As
shown in Fig. 2B, the biggest discovery was that over 80% of the
Ru was found in the cytoskeletal fraction for cells treated with
RPC2. To the best of our knowledge, there are no known
interactions between RPCs and the cytoskeleton. Moreover, this
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Ru content in whole (top) and fractionated (bottom) H358 cells.
(A) Mass of ruthenium in ng per million cells for H358 cells incubated
with 20 mM of RPC1 or RPC2 for 12 h at 37 C or 4 C. (B) Percent
ruthenium found in four different fractions of H358 cells (nucleus,
cytosol, mito/Golgi/ER, cytoskeleton). The cells were fractionated
using a QIAGEN Compartment Kit and Ru ion content was analyzed
using ICP-MS. The total amount of RPC 1 in the fractionated cells was
2.0 ng per million cells and RPC2 15 ng per million cells.
Fig. 2
heavily skewed distribution is not seen for RPC1, indicating
selective binding between RPC2 and some cytoskeletal proteins.
RPC2 induces changes in cellular morphology and MT
structure in xed cells
As shown in Table 1, the two RPCs under study have substantially differing levels of cytotoxic activity. RPC1 is modestly
cytotoxic with IC50 values are greater than 50 mM for the two
malignant (MCF7, H358) and two non-malignant (MCF10,
HUVEC) cell lines examined.
In contrast, RPC2 showed high cytotoxicity with low micromolar IC50 values against all four cell lines. For comparison, the
IC50 values for nocodazole and paclitaxel are included in Table
1, both show low micromolar to nanomolar cytotoxicity.
Table 1 The cytotoxicity IC50 of RPC1, RPC2, PTX, and NCZ against
various cell lines
Compounds IC50 (mM)
Cell line
RPC1
RPC2
NCZ
PTX
H358
MCF7
MCF10
HUVEC
86.7 4.1a
>50
>50
92
1.7 0.1
1.5 0.3
1.5 0.5
2.8 5.1
1.0
3.2
0.48d
0.2d
0.1b
0.025c
0.2c
8.2 104e
a
Ref. 39. b Ref. 40. c Ref. 41. d Ref. 42. e Ref. 43.
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To determine if RPC2 affected the cytoskeleton in cells, MTs
were examined in MCF7, H358, and HUVEC using immunouorescent labeling techniques and confocal uorescent microscopy. The control cells (vehicle) showed that the MT structure is
comprised of ne laments spread throughout the cytoplasm
(Fig. 3, rst column). In these images, the MT's (anti-tubulin) are
shown in green and the cell nucleus in red (propidium iodide,
PI). Cells treated with the IC50 dose of RPC2 for 12 h revealed
a unique response to treatment (Fig. 3, second column). The
H358 cells show dramatic changes with a collapse of the cell
around the nucleus. The MTs became less organized and more
condensed, and several cells displayed pyknotic nuclei, indicative
of apoptosis. The MCF7 cells reveal little change in cell structure
at this dose and time interval, although several nuclei appear in
the early stages of DNA condensation. The HUVEC cells show
large amounts of MT condensation, which appeared as Furinfoci
or blobs at the edge of the cell membrane.
We compared these changes with those for known MDAs and
MSAs and examined the cell's appearance aer treatment with
NCZ (IC50 dose) and PTX (IC50 dose), respectively. In the third
column of Fig. 3, all three cell types became pyknotic upon
treatment with NCZ, although each appeared in a different stage
of nuclear degradation, indicating different temporal responses
to the drug. Notably, there was not much MT structure evident,
nor any bundles of MTs. PTX treatment also resulted in apoptotic
cells; however, the MTs were clearly observed as bundles (MCF7
and H358) or as rounded blobs (HUVEC and H358; Fig. 3). In
addition, several H358 and MCF7 cells showed extensive nuclear
fragmentation indicating apoptosis (Fig. 3, second column). The
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MCF7 cells displayed elegant examples of MT bundles, organized
at specic microtubule organizing centers in the cell, but somewhat abnormal as the bundles were thick and not dispersed
evenly throughout the cell. Some cells were apoptotic with fragmented and condensed nuclei. The nuclei showed MT asters,
including one in apparent metaphase, although oddly, the
nuclear material appeared to be at the edge of the cell. The
HUVEC cells treated with PTX showed the least perturbation
from the control, but were nonetheless augmented.
Comparison of cells treated with RPC2 with those treated with
NCZ or PTX is not straightforward as some changes resemble
traits exhibited by NCZ and others, PTX treated cells. Clearly
there were differences in changes with cell type, with H358
showing the most dramatic variations with all three drugs. MCF7
responds strongly to NCZ and PTX, but not to RPC2. HUVEC cells
responded most strongly to RPC2 then NCZ and lastly PTX
(Fig. 3). The formation of large foci of MTs (green blobs) in the
H358 and HUVEC lines for RPC2 suggests the compound is
acting to condense and stabilize MTs. A western blot experiment
analyzing the tubulin content in the cytosol of H358 cells treated
with PTX (0.1 mM) or RPC2 (1.7 mM) for 12 h before harvesting
and lysis, shows that both agents act to substantially reduce the
amount of free tubulin present the cytosol (see Fig. 4). This is
interpreted as being due to the bulk of the free tubulin becoming
sequestered in MTs due to the MSA activity.44
RPC2 inhibits MT assembly in live cells
To see how RPC2 affects MT dynamics, live MCF7 and MCF10a
cells were examined expressing a microtubule plus-end binding
Fig. 3 Fluorescent microscopic images of fixed cells immunostained for tubulin (green) and the nucleus (propidium iodide, red) after treatment
with RPC2, NCZ, or PTX for 12 h. First row: H358 cells treated without (control), and with 1.7 mM RPC2, 1.0 mM NCZ, 0.1 mM PTX. Second row:
MCF7 cells treated without (control), and with the 1.5 mM RPC2 for 12 h, 3.2 mM NCZ, and 0.025 mM PTX for 12 h. Third row: HUVEC cells treated
without (control), and with 2.8 mM RPC2, 0.2 mM NCZ, and 0.82 nM PTX for 12 h. Concentrations used were the IC50 for that cell line. The atubulin antibody was coupled with Alexa Fluor 488-conjugated goat anti-mouse igG (H + L) secondary antibody.
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Fig. 4 Western blot analysis of microtubule polymers (a-tubulin)
isolated from H358 cells treated with the IC50 of PTX (0.1 mM), and
RPC2 (1.7 mM) for 12 h. The Ponceau staining shows equal protein
loading.
protein (end-binding protein 3; EB3) that is fused to a green
uorescent protein fragment (GFP-EB3) using spinning disk
confocal uorescent microscopy. Untreated cells revealed that
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the growing plus-ends of the MTs moved as green ‘comets’
demonstrating both a bright head and a lingering tail. A movie
of these comets in an control cell shows the comets organized
together in different parts of the cell, with groups of comets
travelling in the same net direction or radiating out from
various MT organizing centers (MOCs) at varying places, as
others have shown previously (e.g. see Movie S1†).45–48
Fig. 5 shows uorescent images collected for control and
RCF2-treated MCF7 and MCF10a cells at ve time points (before
treatment, 30 min, 1 h, 2 h, and 3 h post treatment) upon
incubation with the IC50 dose of RPC2 or PTX. Each image has
a corresponding time-lapse movie, capturing at 3 to 10 s intervals, in which the movement or lack of movement of the comets
was observed (see ESI Movies S1–S20,† all of which are
embedded in ESI Fig. S1†).
Changes were apparent at the 1 h time point for cells treated
with RPC2 and 30 min for the PTX treated cells with effects for
both more pronounced at longer time periods. For both RPC2
Fig. 5 Spinning disk confocal fluorescent microscopic images of live MCF7 and MCF10a cells expressing the GFP-EB. Images show the initial
frame of a short movie (4–9 s) and changes between images are due to the longer incubation period with 1.5 mM RPC2 or 0.1 mM PTX for MCF7
and 0.2 mM PTX for MCF10a. A figure in which each frame is a clickable movie in the same format as given here is provided in the ESI (Fig. S1†).
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These effects are more pronounced at longer time periods. MT
function is better preserved in the MCF10a cells over the MCF7
cells, even at longer incubation times, suggesting less sensitivity
to RPC2 by MCF10a. However, this is not supported by the
cytotoxicity data which has the IC50 for both lines at 1.5 mM.
Presumably, this is an effect of the relatively short time period
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and PTX, the comet's motion is retarded or stopped. The
distinctive MT tracks are eventually lost and the signal disperses
throughout the cytoplasm. In MCF7 cells treated with RPC2, the
number of total comets decreases and those remaining become
larger and less motile. Additionally, MT growth becomes
disorganized with no clear net directional motion observed.
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Fig. 6 RPC2 affects the cell cycle populations in MCF7 breast cancer cells. MCF7 cells were untreated (A) or treated with 1.5 mM (B) and 15 mM (C)
of RPC2 for 12 h, fixed with 66% ethanol, washed in PBS, treated with RNase for 30 min at 37 C and stained with PI. Percent population: (A) sub G
7%, G1 78%, S 5%, G2/M 9%; (B) sub G 5%, G1 81%, S 6.3%, G2/M 7.8% (C) sub G 18%, G1 60%, S 8%, G2/M 14%.
270 | Chem. Sci., 2020, 11, 264–275
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aer introduction of the drug (3 h) compared to a typical MTT
assay (96 h). PTX treated MCF10a cells are similarly more
resistant to the effects of treatment over the MCF7 cells, but the
level of MT disruption is greater here than seen with RPC2.
Some of the movies obtained with RPC2 show strong photobleaching of the GFP signal with time. The movies of RPC2treated MCF10a at 30 min, 2 h, and 3 h (Movies S7, S9 and S10),
provide a typical examples of this effect. Initially, well-formed
comets with tails are evident, but these barely move and simply
fade to small round bright spots or disappear altogether as the
movie progresses. Some of this bleaching is the result of the MTs
growing slower, which increases the EB3 dwell time at the plus
end of the MT. In addition, RPC2 is also a well-known luminescent agent (labs 460 nm; lem 640 nm)49 which makes it an
excellent receptor for uorescent resonance energy transfer
(FRET) from the GFP excited state.50 Furthermore, RPC2 is an
excellent photosensitizer for the conversion of triplet oxygen to
singlet oxygen, which can be highly destructive to any adjacent
organic matter.49 Both likely happens with prolonged laser illumination used to obtain movies. The extent of the photobleaching varied and, in many instances, cells were imaged with
little bleaching. To avoid this cell damage, we did not make
multiple movies of any single cell and instead chose a different
cell to image at the 30 min, 1 h, 2 h, and 3 h timepoints.
Chemical Science
RPC2 treatment results in a small increase in cells in G2/M
phase and a large increase in sub G1
Next, we examined the effects of RPC2 on the cell cycle. As
shown in Fig. 6, ow cytometry of MCF7 cells shows a small
dose dependent response for cells treated with RPC2. At a 1.5
mM dose (the IC50), the histogram is essentially unchanged from
the control whereas at the higher dose (15 mM), a clear jump in
the percentage of cells in the sub G1 and G2/M phases is
apparent. Populations of dead apoptotic or necrotic cells appear
in the region prior to the G1 peak in ow cytometry, as the cell
size has shrunk and DNA dye uorescence is generally weakened with the loss of DNA structure.51 The percentage of cells in
the G2/M phase rises from 9% to 14% while the percentage of
sub G1 increased from 7% to 18%. Flow cytometry of H358 cells
gives similar results, with increases of both the sub G1 and G2/M
populations with a 10 dose of RPC2 (see ESI Fig. S2†).
RPC2 act as microtubule stabilizing agents in vitro
In vitro tubulin polymerization assays reveal that RPC2
increases the rate of tubulin polymerization and enhances the
extent of tubulin polymerization (see Fig. 7) relative to the no
drug control. The effect is dose dependent (see ESI, Fig. S3†)
and titrations between 0.1 and 50 mM RPC2 reveal the onset of
this effect at doses as low as 0.1 mM. Not only does RPC2
Fig. 7 Effect of different ligands on tubulin polymerization in vitro. The change in turbidity measured by light transmission at 340 nm. Increasing
turbidity indicated tubulin polymerization upon a temperature jump from 4 C to 37 C in the presence of 1 mM GTP and 10% glycerol in general
tubulin buffer (80 mM PIPES pH 6.9, 2 mM MgCl2, and 0.5 mM EGTA). All runs with added drug were done by addition of enough drug to make
a 10 mM solution of drug with the tubulin (3 mg mL1). The orange plot shows the normal tubulin polymerization growth curve in the absence of
any drug. PTX (black plot) was used as a control to show how a microtubule stabilization agent effects polymerization. NCZ (blue plot) was added
as control to show how a microtubule destabilizing agent effects polymerization. The legend on the bottom shows the color and markers for the
RPC1, and RPC2 treated runs. All plots are an average of three individual experiments. Error bars (0.05 OD) omitted for clarity.
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increase the rate of tubulin polymerization relative to the
control, it increases the overall degree of polymerization seen in
solution at steady-state. Signicantly, tubulin polymerization is
also enhanced upon addition of RPC1 (10 mM), although not to
the same extent as with 10 mM RPC2 (see Fig. 7). In this gure,
the effects of PTX and NCZ on tubulin polymerization are
included for comparison. The in vitro activity of RPC1 suggests
that RPCs in general may have some inherent MSA activity,
however the lesser ability to enter the cells and the more even
distribution throughout the cell (see Fig. 2) show this alone
does not translate to cellular activity. Clearly, the DIP ligand
increases the tubulin binding affinity and indicates that SAR
studies can improve their performance. TEM images of the MTs
formed in the presence of RPC2 (10 mM) are unchanged in
appearance from those of the control or those treated with PTX
(10 mM), whereas only small MT fragments or tubulin dimers
are apparent when treated with NCZ (see ESI Fig. S4†).
RPC2 binds preformed MTs with a binding affinity similar to
that of DTX and does not compete for the DTX binding site
Isothermal titration calorimetry (ITC) was used to measure the
binding thermodynamics of RPC2 with tubulin and MTs.
Reverse titrations (protein introduced into solutions with a xed
ligand concentration) were done as the poor solubility of RPC2
limited its concentration range. For comparison, ITC
measurements of colchicine and docetaxel (DTX) were also
performed. The association constant (K), changes in free energy
(DG), enthalpy (DH), and entropy (TDS) were obtained52,53 are
listed in Table 2 and were obtained by tting a binding isotherm
using the CHASM soware to the raw integrated isotherms
(shown in Fig. S5†). Experiments were either performed starting
at 4 C or 37 C to obtain a population of tubulin in the free,
unpolymerized state (4 C) or polymerized state as MTs (37 C),
as these two structures present a different collection of binding
sites to the ligand.
As seen from Table 2, RPC2 binds to preformed MTs tightly,
with a K of 4.9 106 M1 which is nearly the same as that obtained for DTX binding the preformed MTs (K ¼ 5.5 106 M1)
for DTX binding to preformed MTs. Reported association
constants PTX and DTX with MTs are 10.7 106 M1 and 30.9
106 M1, respectively,54,55 which is in excellent agreement with
our data given that different techniques were used (ITC vs.
competitive binding titrations).56 A binding stoichiometry of
one ligand per tubulin dimer is known for DTX and colchicine,
which was also supported for RPC2 from the inection point of
the isotherms at a 1 : 1 mole ratio. ITC experiments measuring
Table 2
the binding heat of RPC2 to free tubulin showed excessive heat,
consistent with the induction of MT polymerization and preventing any meaningful interpretation of the binding heat. ITC
experiments injecting free tubulin into colchicine produced
insufficient heat to successfully determine the binding
parameters.
The differences in enthalpies and entropies of binding
between RPC2 and DTX show they have very different modes of
binding and are very similar to those seen for colchicine. In
competition experiments, the MT:DTX complex binds RPC2
with the same energetics as MTs do, suggesting RPC2 binds
somewhere beside the taxane-binding domain.8
Discussion
RPC2 binds MTs in vitro and in live cells and this binding
retards or arrests MT growth in a manner similar to that seen
for PTX. As MSA activity is observed for RPC2 in vitro, the
presumption is that the same is occurring in vivo, which is most
strongly supported by the movie data, as the interpretation of
the MT structure in xed cells is oen ambiguous. In MCF7
cells, the fast-moving and starburst pattern of MT comets
characteristic of normal MT plus end growth is replaced by
fewer, but larger comets that move slowly or are completely
arrested by 2 h of treatment. Moreover, the organization of the
MT growth by MOCs becomes less apparent and even absent in
some cases. PTX is known to reduce the free tubulin concentration in cells to below a critical threshold necessary for organization by MOCs,44 and the western blot data in Fig. 4 and
movie data show RPC2 behaves in the same manner. A review of
how simple divalent cations affect MT formation and depolymerization shows that divalent cations, such as Ca2+, Pb2+ and
Hg2+, generally act to depolymerize or destabilize MTs.12,13 The
MSA activity seen for RPC2 contrasts with these simple cations,
and shows the observed effect is not simply a charge effect.
Flow cytometry of MCF7 or H358 cells stained with PI
showed only a small rise in G2/M population upon treatment
with RPC2 and this required a 17 mM dose, as the IC50 dose (1.5
mM) showed no effect. By comparison, the population in G2/M
for PTX (0.25 mM) treated mouse broblast cells is 50% aer
9 h and 100% by 27 h.57 The IC50 for PTX in this cell line is 0.86
mM.58 Apparently, RPC2 is functioning in some other manner
beside the mitotic arrest mechanism common for many MSAs.
G2/M block is not the only mechanism by which MSA's,
including PTX, function as the formation of aberrant MT
structures (typically bundles) throughout the cell cycle; oen
Thermodynamic binding data for RPC2, DTX, and colchicine with tubulin and preformed microtubules as determined by ITC
Complex
Ligand
K (M1) 106
DG (kcal mol1)
DH (kcal mol1)
TDS (kcal mol1)
Tubulin
MT
MT
MT
MT:DTX
DTX
DTX
RPC2
Colchicine
RPC2
8.1
5.5
4.8
5.0
2.0
9.4 1.0
9.2 0.9
9.1 0.9
9.1 0.9
8.6 0.9
28.7 4.1
33.4 6.5
16.6 2.3
12.8 2.4
14.6 7.7
19.2
24.7
7.5
3.7
6.0
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implicated in cell death.59–61 RPC2 has also been shown to
induce rapid depolarization of the mitochondria in A549 cells
(within 2 h of administration), but this activity was not matched
with an equal loss in cell viability, with 44% of the cells still alive
24 h later and the cell death was attributed to necrosis.23 The
disconnect between time of mitochondrial depolarization and
cell death was observed as an oddity by the authors of this
report. Our ndings and Glazers data support that RPC2 was
also acting via microtubule disruption, which alone or in
concert with the mitochondrial effects is responsible for cell
death.
ITC binding data reveals the strength of the binding for
RPC2 is comparable with DTX in preformed MTs and indicates
that the binding site is not the taxane site. The extensive
intracellular colocalization of RPC2 with the cytoskeletal
proteins reveals this selective binding is occurring in the
cellular milieu. Our subcellular localization data conicts with
that reported by Glazer and coworkers in which RPC2 was
shown to co-localize with mitochondria and lysosomes.23 Using
the inherent luminescence of RPC2, confocal luminescence
microscopy experiments with additional organelles specic
dyes, Mitotracker Green or Lysotracker Green, showed colocalization with these two cellular organelles. The phosphorescence
of RPC2 was also used by Audi et al. in microscopy experiments
shows co-localization of RPC2 with the nucleus in MDA-MB231
cells, however some localization at the cell membrane is also
apparent. Part of this discrepancy is likely due to the fact that
microtubules are only tens of nanometers thick and the binding
of RPC2 to them is diffuse through most of the cytoplasm,
making it extremely hard to resolve at the light microscopy level.
Secondly, microtubules are closely associated and more densely
packed around the mitochondria and lysosomes, than for most
other organelles62,63 and therefore part of the apparent
discrepancy in co-localization data (Glazer data supports localization in the mitochondria and lysosomes whereas our data
support co-localization in the cytoskeleton) is due to this
overlap.
To date, there are no reports of RPCs or any coordination
complexes acting selectively on the cytoskeleton in vivo or on
cytoskeletal proteins in vitro. While the potent cytotoxicity of
RPC2 has been known for decades,21–23 the mechanism of action
was either unknown or attributed to mitochondrial poisoning.
Other cytotoxic RPCs are known and more are being developed
as the eld grows, but until this report the only cellular targets
implicated have been the nuclear DNA, mitochondria, cell
membrane, endoplasmic reticulum, and ribosomes.30,32,64,65
While not all RPCs share the three-bladed propeller structural
motif, this trischelate structure is the most commonly explored
motif to date in terms of biological probes, cellular interactions,
and cytotoxicity.
From our cellular uptake data, we can infer that majority of
the cytotoxicity of RPC2 over RPC1 is due to the enhanced
uptake (approximately 10 fold greater) by the more lipophilic
complex (log P 1.4 and 1.5, respectively66). The differential
levels of colocalization with the cytoskeletal proteins, 82% for
RPC2 and 23% RPC1, reveals that the necessity of the DIP ligand
over phenanthroline for tight binding. It is unknown if one, two
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Chemical Science
or all three ligands must be DIP, but a review of the cytotoxicity
of seven other RPCs containing at least 2 DIP ligands, i.e.
[(DIP)2Ru(L-L)]2+ (where L-L is a chelating diamine), show IC50's
ranging from 4 to 11 mM against B16, SF, ML2, 4T1 or MCF7 cell
lines.20,21,67 This high potency is suggestive that these other
RPCs are also acting as MSAs in cells and the ‘Ru(DIP)2’ fragment is all that is needed for activity. We do not observe
signicant differences in the IC50's for malignant versus nonmalignant cell lines for RPC2 and most other RPCs with
a ‘Ru(DIP)2’ fragment behave similarly (when such data is
available). Two important exceptions are [(DIP)2Ru(tatpp)]2+
and [(DIP)2Ru(tatpp)Ru(DIP)2]2+ (tatpp ¼ tetraazatetrapyridopentacene), which show selectivity indices (SI ¼ IC50 MCF10/
IC50 MCF7) of 71 and 11, respectively.20
There are no obvious correlations between the structure of
RPC2 and the structures of other natural product MSA's (and
derivatives thereof). Natural product MSA's tend to be more
cytotoxic with IC50's in the low micromolar to nanomolar
regime where as RPC2 is in the low micromolar regime. Most of
the natural products exhibit very complex organic structures
containing multiple fused rings, numerous chiral centers, and
require challenging multi-step syntheses.68 Attractively, RPCs
are relatively simple and can be prepared in one to three steps
from the reaction of the commercially available ligands with
a Ru salt.22,24,25,69,70 We prepared over 2 g of RPC2 using two steps
by reuxing RuCl3 and excess DIP in ethanol, isolating the
hexauorophosphate salt and metathesis to the chloride salt.
Overall yield was 83% and it took less than 2 days. RPC2 has
recently become commercially available (e.g. Sigma-Aldrich,
Alfa Aesar, Spectrum Chemical), as a uorescent dye. It
should be noted that RPC2 is chiral at the metal ion (D3 point
group symmetry) and is typically isolated as the racemate, but
can be resolved to stable enantiomers.71 This newly reported
MSA activity for RPC2 and their relative ease of preparation and
derivatization suggests this class of compounds are ripe for
exploration in this new application.
Conclusions
The combination of the live cell movies, xed cell MT structure,
cytotoxicity, sub-cellular localization, in vitro MT binding,
polymerization, and TEM data build a convincing case that
RPC2 enters cells, binds, and disrupts the normal MT network
by stabilizing the MTs present. RPC2 exhibits a reasonably high
binding constant with preformed MTs which does not appear to
be the taxane binding site favored by many MSAs. In vitro
polymerization assays show RPC1 can also promote MT
formation, suggesting that RPCs in general can act as MSAs (to
different degrees) and that some of the DIP containing RPCs,
previously explored as cytotoxins are actually targeting MTs.
This is the rst report of any transition metal complex or
organometallic complex demonstrating MT binding and
disruption of MT function. While much remains to be elucidated about the MSA activity of this and related compounds,
these data show that RPCs with proper ligand modications are
likely to constitute a new, synthetically accessible, class of
Chem. Sci., 2020, 11, 264–275 | 273
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compounds that can be used to target MTs in cells, with
potentially therapeutic benets in vivo.
Conflicts of interest
There are no conicts to declare.
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Acknowledgements
We thank the Robert A. Welch Foundation Y-1933-20170325
(F. M. M.) and W. W. Smith Charitable Trust (C. J.) for nancial support.
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