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A Double-Edged Sword: The Anti-Cancer Effects of Emodin by Inhibiting
the Redox-Protective Protein MTH1 and Augmenting ROS in NSCLC
Divya Wahi1, Deepika Soni1, Abhinav Grover1*
From 1School of Biotechnology, Jawaharlal Nehru University, New Delhi, India - 110067.
* Corresponding Author
Running Title:
Anti-Cancer potential of Emodin on the Redox-Protective MTH1
To whom correspondence should be addressed: Dr. Abhinav Grover, School of Biotechnology,
Jawaharlal Nehru University, New Delhi, India - 110067, Telephone: +91-11-26738728; FAX: +91-
11-26702040; E-mail: [email protected]; [email protected]
Author Email: [email protected] , [email protected]
"The authors declare no potential conflicts of interest."
Keywords: Lung Cancer, NCSLC, MTH-1, NUDT-1, ROS, Emodin, oxidative DNA damage.
Abstract
Background
Reactive oxygen species (ROS), playing a two-fold role in tumorigenesis, are responsible for
tumor formation and progression through the induction of genome instability and pro-
oncogenic signaling. The same ROS is toxic to cancer cells at higher levels, oxidizing free
nucleotide precursors (dNTPs) as well as damaging DNA leading to cell senescence.
Research has highlighted the tumor cell-specific expression of a redox-protective
phosphatase, MutT homolog 1 (MTH1), that performs the enzymatic conversion of oxidized
nucleotides (like 8-oxo-dGTP) to their corresponding monophosphates, up-regulated in
numerous cancers, circumventing their misincorporation into the genomic DNA and
preventing damage and cell death.
Methods
To identify novel natural small molecular inhibitors of MTH1 to be used as cancer
therapeutic agents, molecular screening for MTH1 active site binders was performed from
natural small molecular libraries. Emodin was identified as a lead compound for MTH1
active site functional inhibition and its action on MTH1 inhibition was validated on non-
small cell lung cancer cellular models (NSCLC).
Results
Our study provides strong evidence that emodin mediated MTH1 inhibition impaired NSCLC
cell growth, inducing senescence. Emodin treatment enhanced the cellular ROS burdens, on
one hand, damaged dNTP pools and inhibited MTH1 function on the other. Our work on
emodin indicates that ROS is the key driver of cancer cell-specific increased DNA damage
and apoptosis upon MTH1 inhibition. Consequently, we observed a time-dependent increase
in NSCL cancer cell susceptibility to oxidative stress with emodin treatment.
Conclusions
Based on our data, the anti-cancer effects of emodin as an MTH1 inhibitor have clinical
potential as a single agent capable of functioning as a ROS inducer and simultaneous blocker
of dNTP pool sanitation in the treatment of NSCL cancers. Collectively, our results have
identified, for the first time, that the potential molecular mechanism of emodin function,
increasing DNA damage and apoptosis in cancer cells, is via MTH1 inhibition.
Introduction
Currently, worldwide statistics reveal lung carcinoma is the chief reason for cancer related
deaths [1]. The most widespread type of lung cancer, non-small cell lung cancer (NSCLC) is
difficult to treat owing to its relative insensitivity to chemotherapeutics and radiotherapy and
its low five-year survival rate [2, 3]. Despite several successful advances on the mechanistic
insights into lung cancer development, the concise molecular pathways causing mutations
leading to carcinogenesis are still being elucidated. Studies note that cellular environment
stresses, such as reactive oxygen species (ROS) causing oxidative stress, are key contributors
to lung cancer pathogenesis [4-6]. In addition to the generation of ROS as a routine by-
product of the respiratory chain’s cellular metabolic process, certain exposures of the cellular
environment, such as tobacco smoke, are responsible for the increase in ROS levels of normal
cells [4, 7].
ROS is responsible for the oxidation of cellular pools of nucleotide precursors and protein
building blocks, inducing multilevel genetic mutations leading to cell senescence [8].
Causing the oxidation of precursor nucleotide moieties and the nucleic acid material itself,
ROS leads to the production of 8-oxo-guanine (8-oxoG) in the nucleotide pool via direct
oxidation of dGTP or DNA guanine bases. Then the DNA polymerases often insert oxidized
8-oxo-dGTP that pair opposite to the adenine or cytosine bases during DNA replication,
causing an oxidized base lesion of G to T transversional mutations [9].
The gradual cellular accumulation of such nucleic material lesions induces increased cellular
dysfunction, spontaneous tumorigenesis susceptibility, and eventually carcinogenesis. To
tackle such deleterious cellular outcomes, human cells equip themselves with enzymes to
sanitize the oxidized nucleotide pools. The 18kD human MutT homologue 1 (MTH1) protein,
also known as the Nudix hydrolase 1 (NUDT1), is the predominant pyrophosphatase that
functions to hydrolyze the oxidized purine nucleotide bases at the cellular level leading to
their degradation [10, 11]. MTH1 hydrolyses the oxidized purine deoxyribonucleotide bases
such as 8-oxo-dGTP, 2-OH-dATP, and 8-oxo-dATP, along with their corresponding
ribonucleotide analogues to their monophosphate equivalent forms [12]. This enzymatic
function of MTH1 protein prevents the incorporation of oxidized nucleotide precursors into
the rapidly proliferating genetic material [13, 14].
Although MTH1 has a crucial cellular role, germline MTH1 knockout mice are
developmentally normal and the treatment of such mice with ROS generating chemicals
increases DNA oxidation and cellular toxicity [15-17]. This implies MTH1 genetic
knockdowns and small molecule inhibition leads to tumor specific DNA damage and
senescence both at the in vitro and in vivo levels, while showing minimal outcomes on the
normal cells having lower ROS levels. This research emphasizes that the MTH1 function
becomes significant in biological conditions where sustaining elevated levels of ROS
increases the oxidized nucleotide pool burdens. Thus, MTH1 function becomes crucial to
cellular environments with increased ROS.
As studies have stated MTH1 is abundantly expressed in numerous cancer types, such as
lung, brain, and gastric cancers. They are regularly exposed to increased levels of oxidative
stress compared to the adjacent normal cells, indicating there is a significant MTH1
contribution to tumor cell proliferation [18-20]. Research indicates MTH1 is indeed
selectively essential in NSCLC cells to support uninterrupted cell proliferation and maintain
genome integrity [21]. Also, xenograft studies show that MTH1 inhibition resulted in
decreased tumor proliferation [22, 23]. MTH1 mRNA overexpression is observed in cancer
cells and has an inverse correlation with 8-oxo-dG levels in the cellular milieu [18].
Researchers have also observed an increased MTH1 function in NSCLC in comparison to
normal lung tissue. Additionally, the blocking of MTH1 function contributes to epithelial-
mesenchymal transition and cell proliferation suppression, revealing MTH1 as a promising
therapeutic target, particularly in NSCLC cases [24, 25].
Owing to their rapid proliferation and growth, cancer cells tend to re-organize their cellular
metabolic pathways. Consequently, they are under continuous exposure to elevated ROS
levels leading to 8-oxo-GTP accumulation and eventual cell death [26, 27]. Studies state
robust MTH1 functionality is essential for avoiding the oxidative stress laden hypermetabolic
tumors that undergo rapid cell proliferation [28]. Thus, cancer cells gain function, acquiring
specific mechanisms that prevent 8-oxoG base accumulation in the DNA. The significance of
MTH1 enzyme function in cancer cell survival is currently a highly controversial topic and
the focus of intense research. Recently researchers have identified chemical inhibitors to
MTH1 that show anti-cancer function both in vitro and in vivo [22, 23]. However, the
identification of MTH1 inhibitors of natural origin has yet to be accomplished. The rising
need for therapeutic interventions in cancer progression is an emerging field in cancer
management via the identification of phytochemicals that are functionally non-toxic, cost-
effective as drug candidates, and physiologically bioavailable in the organism. In the present
study, we screened MTH1 inhibitors from natural small molecule compound libraries and
identified emodin as a leading novel natural MTH1 inhibitor. We also highlight the dual
function of emodin leading to cancer cell senescence; it can cause increased ROS in NSCLC
cells along with MTH1 function inhibition. These findings are in line with previous works,
indicating emodin causes increased ROS in cancer cells but the exact mechanism of how
ROS augmentation led to apoptosis in cancers remained un-elucidated until now [29-31].
Emodin (1, 3, 8-trihydroxy-6-methylanthraquinone) is an ancient natural hydroxy-
anthraquinone that is isolated from the roots and rhizomes of a number of medicinal plants
(frangula, rhubarb, etc.) and exhibits diverse biological effects such as being antibacterial,
anti-inflammatory, immunosuppressive, vasorelaxant, and diuretic [32-36]. Recently, emodin
gained special attention as in vitro and in vivo research stressed its anti-cancer effects through
multiple mechanisms in several types of malignancies by inhibiting cancer cell growth,
increasing ROS levels, chemotherapeutic sensitization, and apoptosis induction [29, 37-40].
The existing literature on emodin has primarily focused on the direct toxicity it exerts on
cancer; however the precise underlying molecular pathway is still to be elucidated.
In previously conducted studies, it has been reported that emodin exerts apoptotic effects on
various human cancer cell lines, including NSCLC subtypes, but the effects of emodin on
NCI-H-520 cells has remained unexplored. Thus, our research examines for the first time the
effects of emodin on the NCI-H-520 cell line model. The recent resurgence of research on the
anti-cancer potential of emodin has shown great advances and is found to have anti-tumor
effects on various types of cancers with diverse mechanisms. Thus, the exact mechanism of
emodin requires further study.
As per our extensive search, the current study is the first to demonstrate the association
between emodin causing increased ROS and simultaneous MTH1 functional inhibition,
acting as a double-edged sword against cancer. We screened MTH1 inhibitors from natural
small molecule compound libraries and identified emodin as a lead novel natural MTH1
inhibitor. Although some works state emodin treatment has been responsible for increased
ROS and subsequent DNA damage, this is the first study to directly show a correlation
between emodin mediated MTH1 activity inhibition using molecular docking techniques and
biophysical analysis; this leads to increasing DNA damage and apoptosis in cancers. The
present study highlights the combined effects of increasing ROS and the simultaneous
blocking of dNTP pool sanitation by MTH1 on human non-small cell lung cancer cell line
models.
Materials and Methods
Materials
Roswell Park Memorial Institute (RPMI) medium containing phenol red, fetal bovine serum
(FBS), and penicillin-streptomycin antibiotics (200 units/ml) were obtained from Gibco BRL
(Grand Island, NY, USA). The 96-, 24-, and 6-well plates and T25 and T75 cell culture flasks
were obtained from NUNC (Roskilde, Denmark). Dimethyl sulfoxide (DMSO) was procured
from Merck-Millipore (NJ, USA). The emodin (E7881), and 3-(4, 5-dimethylthiazol-2-yl)-2,
and 5-diphenyltetrazolium bromide (MTT) were procured from Sigma Aldrich (USA). The
JC-1 iodide (sc-364116), 3,3′-dihexyloxacarbocyanine iodide (DIOC6) (sc-205905), and
propidium iodide (sc-3541) were procured from Santa Cruz Biotech (Santa Cruz, CA, USA).
The CellROX™ Green (C10444), CellROX™ Deep Red (C10422), and Hoechst 33342
(H1399) were procured from Thermo Fisher Scientific (USA). The organic solvents were of
HPLC-grade (Merck, NJ, USA). All other chemicals were from Sigma Aldrich (USA) or as
otherwise specified. The list of antibodies used is provided in the supplementary data.
Cells and Cell Culture
The NSCLC cell line models NCI-H-520 (squamous cell carcinoma), NCI-H-460 (large cell
carcinoma), and A-549 (adenocarcinoma) cells were obtained from the cell repository at
National Centre for Cell Science (NCCS, Pune, India). All cells were cultured in RPMI-1640
media (Gibco) supplemented with 10% (v/v) fetal bovine serum (Gibco), 100 μg/ml
streptomycin, and 100 U/ml penicillin (Gibco). The cultures were grown and maintained at
37°C in a humidified incubator containing 5% CO2. The cultures were passaged on reaching
80% confluence and split into a ratio of 1:4 to be used for experiments.
Cell Cycle Analysis
The NSCLC cells were cultured until 80% confluence, and then seeded in 6-well plates in
3ml of the RPMI medium at a cell density of approximately 105 cells/ml. The cells were
subsequently treated with emodin at different dilutions (0, 25, 50, and 75 μM) in DMSO,
(vehicle < 0.5% volume) for 24, 48, and 72 h. Post-treatment, the cells were harvested by
trypsinization and washed with PBS. The cells were then fixed using ice-cold 70% ethanol
for 24 h at 4°C. The samples were then treated with 20 μg/ml RNase A and 50 μg/ml PI
(propidium iodide) for 30 min. Then, the percent cell populations in the G0/G1, S, and G2/M
phases were acquired by flow cytometry (BD FACSAria Flow Cytometer, Becton Dickinson
Biosciences, USA). A small volume of cells without drug treatment were used as an
unstained control.
Mitochondrial Membrane Potential (ΔΨM) Assay
JC-1 and DIOC6 [3] Iodide: Fluorometric Assessment of Mitochondrial Membrane Potential
The NSCLC cells were cultured until 80% confluence, and then seeded in 96-well plates in
100 μL of the RPMI medium at a cell density of approximately 103 cells/well. The cells were
treated with emodin at different dilutions ( 0, 1, 10, 25, 50, 75, 100, and 200 μM ) in DMSO
(vehicle < 0.5% volume) for 24, 48, and 72 h. Post-treatment, the media was aspirated and
100 µl of fresh media containing 1µg/ml JC-1 (working concentration from 1mg/ml JC-1 in
DMSO stock solution) or 1µM DIOC6 [3] iodide (working concentration from 1mM DIOC6
[3] iodide in DMSO stock solution) was added to each well. The 96-well plate was incubated
at 37°C in a humidified 5% CO2 chamber for 20 min. After incubation, the JC-1 dye
fluorescence was acquired with a 96-well ELISA plate-reader (2300 EnSpire Multimode
Plate Reader, PerkinElmer, MA, USA) using wavelengths of excitation at 488 nm, emission
at 530 nm for monomers and excitation at 540 nm, and emission at 590 nm for J-aggregates.
Annexin V/PI Apoptosis Analysis
Flow Cytometric Assessment of Apoptosis
The NSCLC cells were cultured until 80% confluence, and then seeded in 6-well plates in 3
ml of the RPMI medium at a cell density of approximately 105 cells/ml. The cells were
subsequently treated with emodin at different dilutions (0, 25, 50, and 75 μM) in DMSO
(vehicle < 0.5% volume) for 24, 48, and 72 h. Post-treatment, the cells were harvested by
trypsinization and washed with PBS. The cells were then stained with 5 µL Alexa Fluor® 488
annexin V and 1 µL 100 µg/mL propidium iodide (PI) working solution per 100 µL cell
sample in the dark at room temperature for 15 min, according to the manufacturer’s
instructions (Dead Cell Apoptosis Kit with Annexin V Alexa Fluor™ 488 & PI, Thermo
Fisher Scientific, USA). After incubation the cells were kept on ice. Immediately, the
apoptotic cells were determined by flow cytometry (BD FACSAria Flow Cytometer, Becton
Dickinson Biosciences, USA) acquiring fluorescence emission at 530 nm and >575 nm. A
small volume of cells without drug treatment were used as an unstained control.
Western Blot
The proteins of the total cell lysate were estimated using the Pierce™ BCA Protein Assay Kit
(Thermo Fisher Scientific, USA). Equal volumes of protein were loaded in each well and
separated on 6%-12% SDS-PAGE gels using the protein electrophoresis Western blotting
assembly (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. The
proteins were electrophoresed at 60V in a stacking gel and 100 V in a resolving gel. After
separation, they were transferred onto nitrocellulose membranes (Merck-Millipore, NJ, USA)
followed by blocking with 3% skimmed milk in tris buffered saline-0.1% Tween-20 (TBS-T)
at room temperature for 2 h. The blots were probed with different primary antibodies at their
specific dilutions (1:200-1:1000 in 3% BSA in TBS-T) at 4˚C overnight and then washed in
TBS-T. Horseradish peroxidase-conjugated secondary antibodies (1:2000) at room
temperature for 1 h and then developed using the Immobilon Western Chemiluminescent
HRP Substrate (Merck-Millipore, NJ, USA).
Modified Alkaline Comet Assay
Alkaline single-cell gel electrophoresis was performed on NSCLC cells to assess the DNA
damaging potential of emodin. The cells were seeded in 6-well plates at a concentration of
approximately 105 cells/well and treated with emodin at different dilutions (0, 25, 50, and 75
μM) in DMSO (vehicle < 0.5% volume) for 24, 48, and 72 h. After treatment, the cells were
centrifuged, harvested, and washed with PBS. Immediately, they were resuspended in a
volume of 100 μL low-melting agarose (LMA) and spread on the surface of frosted slides
coated with 1% normal melting agar (NMA) and a coverslip was placed on top of the
mixture. The slides were kept on ice for 10 min before removing the coverslips. The cells
were lysed by incubating the slides in a lysis buffer (2.5 M NaCl, 0.1 M EDTA, 10 mM Tris,
pH 10, 1% Triton X-100, 10% DMSO) for 1 h and treated with an alkaline buffer (0.3 M
NaCl, 1 mM EDTA) for 40 min in the dark. The slides were then immersed in the
electrophoretic buffer (300 mM NaOH, 1 mM EDTA), electrophoresed at 22 V and 300 mA
for 30 min and immediately neutralized using the neutralization buffer (0.4M Tris-HCl, pH
7.5). The slides were stained with ethidium bromide and analyzed on a Nikon real time laser
scanning confocal microscope at 60X and 20X magnification with supporting software. A set
of 100 comets were analyzed in triplicate from each sample set, including the vehicle control.
The tail moment was then calculated as % DNA in the comet tail × tail length, using Andor
Komet version 7.1 software.
Terminal Transferase dUTP Nick End Labeling (TUNEL) Assay
For confocal microscopy based determination of cells exhibiting DNA fragmentation using
TUNEL assay (according to a modified version of the manufacturer's recommended protocol,
DeadEnd™ Fluorometric TUNEL System Kit, Promega, WI, USA), the NSCLC cells were
cultured until 80% confluence and seeded on coverslips, placed in 24-well plates at a cell
density of approximately 104 cells/well. The cells were treated with emodin at different
dilutions (0, 25, 50, and 75 μM) in DMSO (vehicle < 0.5% volume) for 24, 48, and 72 h;
following which the cells were washed (twice) with PBS and fixed with 4%
paraformaldehyde in PBS for 20 min. The fixed cells were then rinsed with PBS and
permeabilized using 0.5% TritonX-100 for 5 min.
The PBS washed slides were incubated for 10 min with an equilibration buffer. The cells on
the coverslips were subsequently incubated with the incubation buffer having a mix of the
equilibration buffer, nucleotide mix, and rTdT enzyme in each well, in a dark humidified
chamber at 37°C for 1 h. Post-incubation, the cells were treated with 2X SSC buffer for 15
min at room temperature to stop the reaction and a PBS wash was performed. The DNA was
counterstained with a 2 µg/ml working Hoechst staining solution from a Hoechst 10 mg/mL
stock in PBS for 10 min at room temperature and the coverslips were mounted in DABCO
(Sigma Aldrich) mounting medium (containing 1% DABCO [w/v] in 80% glycerol and 20%
PBS). The cells were visualized using a Nikon real time laser scanning confocal microscope
at 60X magnification with supporting software.
Surface Plasmon Resonance (SPR) Analysis
Using a monolayer surface of self-assembled 11-mercaptoundecanoic acid (MUA) on a gold
surface, the Autolab Esprit Instrument (Eco Chemie BV, Netherlands) was activated with N-
hydroxysuccinimide (NHS, 0.05 M) / N-ethyl-N-(diethylaminopropyl) carbodiimide (EDC,
0.2 M) for 600 seconds. Subsequently, MTH1 as a ligand was immobilized to the activated
sensor surface for 600 seconds at a concentration of 30 μg/ml and dissolved in the
immobilization buffer (20 mM sodium acetate buffer [pH 5.0], 0.22 μm filtered and
degassed). After MTH1 ligand immobilization, the gold surface was blocked for 200 seconds
with 100 mM ethanolamine (pH 8.5), followed by regeneration for 300 seconds using 50 mM
NaOH (0.22 μm filtered and degassed). Then, the kinetic analysis was performed by flowing
different concentrations of the analyte emodin (1 mM to 25 mM) on the immobilized protein
MTH1 in a running buffer (20 mM sodium phosphate buffer (pH 7.4), 0.22 μm filtered and
degassed) at 25°C. The parameters of association kinetics were monitored for 300 seconds
followed by dissociation kinetics for 100 seconds. After each cycle of association and
dissociation the binding surface was regenerated using 50 mM NaOH and a baseline of 120
seconds was run. All the binding kinetic experiments were performed in triplicate on three
separate sensor chips on the Autolab SPR apparatus using emodin as an analyte and (S)-
crizotinib as the positive control. The results in the form of reference-subtracted sensorgrams
were analyzed using the Autolab equipment software. The binding constant KD was
calculated as KD/KA and plotted as a differential response of emodin binding to MTH1.
8-OHdG estimation
The 8-OHdG DNA was extracted from NSCLC cells using the commercial kit 8-oxo-dG
ELISA kit (R&D Systems, Inc. MN, USA) according to the manufacturer's instructions. The
absorbance was read at 450 nm.
Densitometry and Statistical Analysis
The western blotting bands were scanned using the Syngene G:Box Gel Doc system and the
mean density was analyzed using ImageJ software (version 1.48v, NIH, USA). Densitometry
data is represented as “fold change” as compared with respective vehicle DMSO control,
after normalization with respective loading controls (β-actin).
All measurements were carried out in triplicate, with error bars representing the respective
standard deviation occurring at each data point. The significance of all cases was verified
using a two-tailed t-test. The p-value notations were reported as *p < 0.05, **p < 0.01, and
***p < 0.001.
Detailed procedures for Screening of MTH-1 inhibitors, Molecular dynamics, Assessment of
cell morphology, Assay of cell growth and Viability, Wound- healing Assay, Mitochondrial
membrane potential (ΔΨM) assay, Nuclear morphological Assessment, Anenexin V/PI
Apoptosis Analysis, Cell lysate Preparation, Measurement of intracellular ROS, DNA
Fragmentation Analysis, Indirect Immunofluorescence, Bacterial Expression and purification
of MTH-1, Circular Dichroism Spectroscopy analyses are provided in the supplementary
data.
Results
Virtual Screening and Docking Studies to Identify MTH1 Binding Compounds
A virtual screening of the Sigma natural compound database and the Natural Remedies
natural compound database against MTH1 protein was carried out. The active site of MTH1
protein is composed of the residues Leu9, Phe27, Phe72, Met81, Val83, Trp117, Trp123, and
Phe139 [41]. To screen for MTH1 abrogators, the active site residues along with Asn33,
Asp119, and Asp120 were used for grid generation [41, 42].
The top results based on Glide HTVS docking scores, fulfilling Lipinski’s rule, were selected
for next level filtering and screening using the stringent docking protocol XP. All the
compounds that achieved Glide HTVS scores greater than -7 Kcal/mol were subjected to XP
docking, after which the top three natural compounds showing the highest affinity
interactions with MTH1 (ligand_71308639 taxifolin hydrate [Sigma], ligand_148 quercetin
dihydrate [Natural Remedies], and ligand_3220 emodin [Sigma]) were selected. Taxifolin
hydrate, quercetin dihydrate and emodin had strong binding interactions with MTH1 as
shown by their respective Glide scores of -8.76 Kcal/mol, -8.74 Kcal/mol, and -7.63
Kcal/mol, respectively (Figure 1). All three natural compounds occupied the targeted active
site in the MTH1 structure blocking the oxidized dNTPs’ access into the MTH1 cavity for
interaction. Table 1 depicts the docking result in terms of the Glide score from XP, the Glide
energy, the major interacting residues, and the H-bond distances formed.
However, after reconfirmation of the docking scores with the AutoDock version 4.0.1
software [43], emodin was found to be the most stable binding partner at the MTH1 active
site. The docking scores were -6.83 Kcal/mol for Emodin, -5.77 Kcal/mol for taxifolin
hydrate, and -5.81 Kcal/mol for quercetin dihydrate (Table 2). Further, studies in the
literature noted previous links between ROS, DNA damage, and emodin [29, 38, 44-46]; thus
emodin was investigated as the lead small molecule natural compound for MTH1 inhibition
studies.
Specificity of Emodin to MTH-1 and Binding Mode Analysis
The Molecular Dynamics (MD) simulation was performed using the emodin-MTH1 complex
docked conformation to analyze the complex conformational dynamics. The MD trajectory
frames of the protein-drug complex were obtained for a 50 ns time scale using the cluster
analysis technique and plotted for post-simulation interactions. The root mean square
fluctuation (RMSD) analysis of the 50 ns trajectory shows an increased RMSD value (> 2nm)
after ~18.5 ns but the deviations remain in the overall range of 0.1 nm until the end of the
simulations. Thus, the RMSD profile was consistently below 0.3 nm for the entire duration of
the simulations and in a time span of 50 ns the emodin-MTH1 complex trajectory achieved
convergence (Figure 2A). The mean residue flexibility of the emodin-MTH1 complex
structure was also investigated using the RMSF analysis. The RMSF score of the emodin-
MTH1 complex had a peak of 0.45 nm for the residues 37 to 44 (Figure 2B).
The statistical analysis of the radius of gyration (Rg) for the emodin-MTH1 complex through
the 50 ns trajectory indicated there is an increased characteristic stability over time to a final
1.41 nm Rg value as compared to the initial 1.46 nm Rg (Figure 2C). The slight decrease in
the Rg score can be interpreted as greater compactness and folding of the protein to make the
drug more available for the initiation of a stable interaction and binding function. The
solvent-accessible surface area (SASA) of the emodin-MTH1 complex stabilizes over a
simulation time of 50 ns (Figure 2D). Decreased SASA values indicate the increasing
hydrophobic surface of the protein highlight there is an increasing fold pattern and relative
stabilization of the emodin-MTH1 structure and decreased intrinsic flexibility, increasing the
likelihood of stable binding of emodin with MTH1.
The emodin-MTH1 structural interactions and intermolecular H-bonding pattern was studied
and the complex stability was described for a simulation duration of 50 ns. The H-bond
lengths decreased with an increase in the simulation time, making the complex more stable,
while initially the H-bond interacting residues had greater bond lengths and weaker
interactions (Figure 2E). Similar to the conclusions from the RMSF analysis, the simulated
emodin-MTH1 complex shows lower flexibility of the conformation and higher stability
owing to the reduced H-bond interactions. Conclusively, the post MD simulation interaction
study highlights the increased stability of the simulated complex in contrast to the initial
complex. The value of the SASA energies are calculated in Table 3.
The Sensitivity of NSCLC Cell Lines to Emodin
To further validate the above molecular dynamic conclusions of docking emodin with MTH1
protein, human NSCLC cell lines NCI-H-520 (squamous cell carcinoma), NCI-H-460 (large
cell carcinoma), and A-549 (adenocarcinoma) were treated with emodin (25, 50, and 75 μM)
or vehicle (DMSO < 0.5% volume) for 24, 48, and 72 h. The overall morphological
assessment of cell health and growth was performed visually and photographed. There was a
marked decrease in cell number and cell growth in a dose and time dependent manner, as the
cells appeared to be more rounded and less adherent to the surface of the flask in the
microscope (Supplementary Figure 1).
The effect of emodin on cell proliferation was then assayed using the trypan blue dye
exclusion assay. The NSCLC cells showed dose and time dependent growth inhibition. In the
NCI-H-460 cells, the total cell number decreased by 77% (p < 0.05) to 62% (p < 0.001) after
48 h and 63% (p < 0.001) to 46% (p < 0.001) following 72 h of emodin treatment compared
to 24 h (Figure 3A). In the A-549 cells, the same emodin concentrations effectively decreased
the total cell numbers by 77% (p < 0.05) to 55% (p < 0.001), 61% (p < 0.05) to 44%
(p<0.001) following 48 and 72 h of emodin treatment compared to 24 h (Figure 3B). In the
NCI-H-520 cells, the range of decrease in total cell numbers was 71% (p < 0.05) to 60% (p <
0.001) and 68% (p < 0.05) to 54% (p < 0.001) following 48 and 72 h of emodin treatment
with respect to 24 h (Figure 3C). We observed a linear increase in the percentage of dead
cells along with the increasing concentrations of emodin with time in all the NSCLC cells,
which was moderate but significant at higher doses (Figure 3).
In the MTT assay, emodin exhibited a dose and time dependent decrease in cell proliferation
as early as 24 h, continuing to 72 h of exposure, with IC50 values of 126, 61, and 18 μM with
increasing time respectively for NCI-H-520 cells (Figure 4A), 97, 67, and 64 μM for NCI-H-
460 cells (Figure 4B) and 124, 76, 54 μM at 24, 48 and 72 h respectively for the A-549 cells
(Figure 4C). Interestingly, the NCI-H-460 cells were the most sensitive, whereas the A-549
were the least sensitive to emodin.
Emodin Inhibited Cell Proliferation and Induced Cell Cycle Arrest
The emodin treatment causes cell cycle arrest in NSCLC cells in a concentration and time
dependent manner. Post-treatment with emodin the cycle of NCI-H-520 cells was altered.
The percentage of cells in the G2/M phase in the emodin treated group was significantly
higher than in the untreated group and the percentage of cells in the S phase emodin group
was significantly lower than in the untreated vehicle control group. At 24, 48, and 72 h G2/M
increased from approximately 19% to 28%, 17% to 37%, and 14% to 29%, respectively, and
the S phase decreased from 37% to 16%, 26% to 21%, and 19% to 14%, respectively (Figure
5A and D).
In the A549 cells, the G2/M phase cells increased in proportion from 14% to 18%, 13% to
22%, and 15% to 20%, respectively, and the S phase cells gradually declined from 17% to
13%, 24% to 17%, and 27 to 17%, respectively, at 24, 48, and 72 h (Figure 5B). In NCI-H-
460, the G2/M cells increased from 11% to 19%, 15% to 20%, and 11% to 15% at 24, 48, and
72 h respectively, and the proportion of the S phase cells decreased from 21% to 8%, 14% to
6%, and 15% to 12%, respectively (Figure 5C).
As binding emodin and MTH1 is demonstrated to induce cell cycle arrest at G2/M and
decrease in the S phase in NSCLC cells, the expression and activation of cell cycle related
proteins was investigated using Western blotting analysis (Figure 5E). While the protein level
expression profile of cyclin-D1, CDK-2, and CDK-4 decreased in NCI-H-460 cells, cyclin-
B1 levels increased on being treated with emodin in a duration dependent manner at 50 μM
compared with untreated vehicle control cells (Figure 5F). This indicates that emodin could
induce apoptosis in NSCLC cells in a duration and dose-dependent manner.
Emodin Induced and Increased the Rate of Apoptosis in NSCLC Cells
Emodin-induced apoptosis in NSCLC cells was apparent by the characteristic features of
apoptosis observed on the Hoechst 33342 staining. We observed cellular surface changes
such as rounding, detachment, and membrane-blebbing as cells prepared to undergo
apoptosis after treatment in a time and concentration dependent manner (Supplementary
Figure 2A). Furthermore, post-nuclear staining with the Hoechst 33342 dye the number of
stressed NSCLC cells increased from < 1 to > 33 in NCI-H-520, < 4 to > 41 in NCI-H-460,
and < 3 to > 35 in the A-549 cell lines with increased treatment (Supplementary Figure 2B).
To verify whether emodin binding to MTH1 induced apoptosis of NSCLC cells, we did a
primary experiment and stained cells with annexin V-FITC and PI, followed by fluorescent
microscopy. The results are presented in Supplementary Figure 3A, where emodin induced a
concentration dependent augmentation in the NCI-H-520 cell apoptosis in 24 h. There is an
increasing green fluorescence (annexin positivity) and red fluorescence (PI positivity) with
increasing emodin treatment. The mean fluorescent intensities are graphically represented in
Supplementary Figure 3B, indicating an increase in the fluorescence signal of apoptotic cells
with increasing emodin treatment for 24 h, as observed under the confocal microscope. The
study concluded a significant apoptosis induction by emodin as observed by fluorescent
microscopy in the NSCLC cells in a concentration dependent manner.
We also performed a confirmatory flow cytometric analysis. As highlighted on the graphs in
Figure 6A-C, only a very small number of apoptotic cells were detected in the vehicle control
group. Based on distinct double-staining patterns in NCI-H-520 treated with emodin, there
was a dose and duration dependent increase in the number of apoptotic cells, with their
population increasing from ~2.5% to ~5.6%, ~3.8% to ~2.9%, and ~5.4% to ~63.9% at 24,
48, and 72 h, respectively (Figure 6D). In the A-549 cells the increase in the number of
apoptotic cells went from ~0.8% to ~3.1%, ~2.9% to ~8.1%, and ~16.5% to ~35.0%,
respectively (Figure 6E), and in NCI-H-460 the increase was from ~0.1% to ~2.3%, ~1.2% to
~8.9%, and ~9.2% to ~44.5% at 24, 48, and 72 h respectively (Figure 6F). These results
indicate emodin-induced apoptosis in NSCLC cells.
As emodin has been demonstrated to induce apoptosis in NSCLC cells, the expression and
activation of apoptosis related proteins was investigated using Western blotting analysis. The
protein level expression profile of Bax (pro-apoptotic protein) and survivin increased and
Bcl-2 (anti-apoptotic protein) decreased in the NCI-H-520 cells treated with emodin in a
duration dependent manner at 50 μM, compared with the untreated vehicle control cells. The
cleavage of caspase-3 and PARP (poly ADP ribose polymerase) revealed that cleaved-
caspase-3 and cleaved-PARP levels increased in emodin treated NCI-H-520 cells compared
with the untreated vehicle control cells (Figure 6G-I). We also observed an increase in the P-
21 protein levels while p-65 NFκβ protein levels decreased with time on emodin treatment
(Supplementary Figure 4A). These results suggest that emodin may activate the caspase-
dependent apoptosis pathway in NSCLC cells.
Emodin Inhibited the Migratory Activity of NSCLC Cells
To evaluate the effect of emodin on cell migration, a wound healing assay was performed on
the NSCLC cell lines. The wound healing assay suggested that emodin suppresses the
migration of NSCLC cells in a dose and duration dependent manner as compared with their
corresponding untreated controls (Figure 7A-C). Treatment with 50 µM emodin visually
inhibited NCI-H-520 cell migration, with wound areas of ~5% in 24, ~17% in 48, and ~57%
at 72 h. The effect of emodin inhibition was ~57% in 24, ~67% in 48, and ~73% at 72 h on
A-549 scratch areas. In NCI-H-460, the cell migration was significantly inhibited with the
wound area ~41% at 24 h, ~70% at 48 h, and ~110% at 72 h (Figure 7D-F).
Given the effects of emodin on NSCLC cell migration, the cell migratory mechanism was
further investigated by Western blot assay. Since integrins and vimentin play an important
role in cancer cell metastasis, the protein expression levels were detected by Western
blotting. In the NCI-H-520 cells, emodin duration dependently reduced the integrin β1 and
vimentin protein expression levels at a concentration of 50 µM in treated cells compared with
the untreated vehicle control (Figure 7G). These data therefore suggest that emodin had an
inhibitory effect on the migration of NSCLC, which was associated with the downregulation
of integrin β1 and vimentin protein expression.
The Effects of Emodin on NSCLC Cells Produced Though Activation of ROS
To verify whether emodin-MTH1 binding caused overproduction of ROS, a primary
investigation was carried out on the amount of intracellular and mitochondrial ROS levels
generated on emodin treatment. The results are presented in Figure 8A-B, where emodin
induced a concentration dependent augmentation in NCI-H-520 ROS in 24 h. There is an
increasing red fluorescence (cellular ROS positivity) and green fluorescence (mitochondrial
and nuclear ROS positivity) with increasing emodin treatment. The mean fluorescent
intensities were graphically represented in Figure 8C-D, with mean fluorescent
intensity/nuclei indicating an increase in the fluorescence signal of ROS in cells with
increasing emodin treatment for 24 h, as observed under the confocal microscope.
Emodin Caused Depletion in the Mitochondrial Membrane Potential (ΔΨm) of NSCLC
Cells
To investigate the possible role of emodin generated ROS in DNA damage and subsequent
apoptosis in NSCLC cells, preliminary fluorescent microscopy and fluorimetry using JC-1
dye was performed. The NCI-H-520 cells were treated with emodin and carbonyl cyanide 3-
chlorophenylhydrazone (CCCP) at 24 h and stained with JC-1 dye. We observed a
concentration dependent increase in the fluorescent intensity ratio of monomer aggregates in
comparison to the vehicle control. The highest fluorescence ratio was observed in cells
treated with protonophore CCCP acting as the positive control (Figure 9A). There was an
increase in the fluorescent intensity ratio of monomer:aggregates from ~0.64 in the vehicle
control to ~0.83 in 75 μM emodin treated cells and ~0.94 in the CCCP control (Figure 9B).
The NSCLC cells were used for fluorometric analysis using JC-1 staining to study the effect
of emodin on mitochondrial membrane potential (∆ψm). A dose and duration dependent
increase in the fluorescent intensity ratio of JC-1 monomer:aggregates were obtained from
fluorimetry. The result indicates that the mitochondrial membrane potential (∆ψm) was
significantly reduced as measured by the increased fluorescence ratios of the JC-1 dye (Figure
9C-E).
To confirm the results of the JC-1 dye, preliminary fluorescent microscopy and fluorimetry
using the DIOC6 dye were also performed. The NCI-H-520 (squamous cell carcinoma) cells
were treated with emodin and CCCP (positive control) and we observed a concentration
dependent decrease in the fluorescent intensities in comparison to the vehicle control. The
lowest fluorescence was observed in cells treated with protonophore CCCP acting as the
positive control (Figure 10A). There was a decrease in the fluorescent intensities from ~17 in
the vehicle control to ~10 in 75 μM emodin treated cells and ~7 in the CCCP control (Figure
10B).
Furthermore, the NSCLC cells were used for fluorometric analysis using DIOC6 staining to
study the effect of emodin on the mitochondrial membrane potential (∆ψm). We observed a
dose and duration dependent decline in the fluorescent intensities and the result indicates that
the mitochondrial membrane potential (∆ψm) was significantly reduced as measured by the
reduced fluorescence of the DIOC6 dye (Figure 10C-E).
The drop in ∆ψm followed a dose and duration dependent pattern and decreased gradually,
confirming that the cellular damage occurring at the mitochondrial level may be co-related to
the apoptotic response of NSCLC cells to emodin. These data support the hypothesis that
ROS generation by emodin causes mitochondrial membrane depolarization and the
subsequent induction of apoptosis in NSCLC cells. It is thus concluded that emodin treatment
led to a dose and duration dependent loss of ∆ ψm.
The Effects of Emodin on NSCLC Cells Produced Though ROS Mediated Accumulation
of Damaged DNA
To further investigate the extent of ROS mediated induction of cell death caused by emodin,
a DNA fragmentation assay was performed using agarose gel electrophoresis. As shown in
Figure 11A, in NCI-H-520 (squamous cell carcinoma) emodin treatment at 24 h did not
generate any DNA fragmentation, but at 48 and 72 h it generated a marked pattern of DNA
fragmentation with a dose dependence, while the vehicle control alone was unable to induce
any DNA degradation. In A-549 (adenocarcinoma) treatment with increasing emodin
concentrations at the 24, 48, and 72 h time points clearly caused DNA fragmentation, which
was similar to the ladder-like pattern observed in NCI-H-460 (large cell carcinoma) (Figure
11B-C).
To further confirm the potentiating effects of emodin on DNA damage in NSCLC cells, a
single cell acrylamide gel electrophoresis (SAGE) assay was performed to be quantitatively
and qualitatively analyzed. The emodin treated cells showed well-formed comets, with tail
lengths increasing in a concentration and duration dependent manner in comparison to the
untreated vehicle controls with no comet like appearance (Figure 12A-C). Post treatment, the
comet tail moments, that is tail lengths of NCI-H-460 in 24, 48, and 72 h, were increased
from approximately 0.1 to 14.4, 1.6 to 25.0, and 2.1 to 68.1, respectively (Figure 12D). In
A549 cells the comet tail moments increased from 0.3 to 24.1, 0.5 to 34.5, and 1.8 to 46.8,
respectively (Figure 12E) and in NCI-H-520 cells, the comet tail moments increased from 0.1
to 10.1, 0.4 to 13.7, and 0.9 to 17 in 24, 48, and 72 h, respectively (Figure 12F). These
observations exhibit emodin-induced NSCLC cell death, which could be mediated through an
apoptotic pathway causing extensive DNA fragmentation and damage.
A TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay was then
performed for the detection of DNA strand breaks induced by emodin treatment. The cells
treated with emodin showed a distinct appearance in nuclear condensation and incorporation
of the Alexa-Fluor labeled nucleotides into the DNA (a positive TUNEL reaction) in a
duration dependent manner compared to the vehicle control (Figure 13A-C). Post-treatment
with emodin, the mean intensity/nuclei of TUNEL reaction positive cells for the NCI-H-520
cells in 24, 48, and 72 h were increased from approximately 1.5 to 4.9, 0.6 to 5.1, and 0.6 to
7.0, respectively. In the A549 cells, the mean intensity/nuclei of TUNEL reaction positive
cells increased evidently with values from 1.3 to 3.3, 2.0 to 5.8, and 1.0 to 10.2 and in the
NCI-H-460 cells, the mean intensity/nuclei increased from 0.7 to 3.9, 0.9 to 4.3, and 0.9 to
5.1 in 24, 48, and 72 h, respectively (Figure 13D). These observations exhibit that emodin
binding to MTH1 increases ROS that eventually induces nuclear DNA fragmentation, a
hallmark of apoptosis in NSCLC cells. Thus, NSCLC cell death is possibly mediated through
an apoptotic pathway causing extensive DNA fragmentation and damage.
Since emodin is known to produce ROS, the relationship between ROS generation and DNA
damage and repair eventually leading to apoptosis requires further understanding. Initial
experiments indicated that emodin induces ROS and subsequent DNA damage that activates
DNA double stranded break repair mechanisms, which fail to repair the DNA owing to
extensive damage caused by emodin mediated MTH1 functional inhibition; thus, leading to
apoptosis in NSCLC cells.
To validate this further, the expression and activation of DNA damage and repair related
proteins was investigated by immunofluorescence labelling followed by confocal microscopy
and Western blotting analysis (Figure 14). Immunofluorescence study of the DNA damage
and repair proteins ATM-p, DNA-PKCs, and 53 BP-1 revealed an increase in NSCLC cells
treated with emodin in a duration dependent manner at 50 μM, compared with untreated
vehicle control cells (Figure 14 A-C).
In addition, to the given the effects of emodin on DNA damage and repair proteins in NSCLC
studied by immunofluorescence, protein levels were further investigated by western blot. The
western blotting analysis of the ATM-p, DNA-PKCs and 53 BP-1 protein expression profiles
revealed similar results. The DNA damage and repair protein expression levels increased in
Emodin treated NSCLC cells compared with untreated vehicle controls (Figure 14 D). These
results suggested that Emodin may activate the double stranded DNA damage and repair
pathway in NSCLC cells.
Expression and Purification of Human MTH1 Protein from E. Coli
To further confirm that emodin and MTH1 binding occurs at the cellular level and is
responsible for the augmentation of ROS, damaged dNTPs, and growing DNA damage in
NSCLC cells, MTH1 protein was studied for its binding to emodin bio-physically. The
plasmid containing the NUDT1 (N-terminal His-tagged) gene was revived from a DH5α
bacterial stab and the NUDT1 plasmid was extracted with the plasmid purity determination
by agarose gel (Supplementary Figure 5A). The plasmid was then transformed into
Escherichia coli BL21-codon plus strain (Figure 15A).
After transformation and production, the protein content of the major purification steps at
each level-the total cell lysate, the lysate supernatant, the flow-through, the wash, the matrix-
bound, and the elution-were analyzed on a 12% sodium dodecyl sulfate polyacrylamide gel
(SDS-PAGE) following Coomassie brilliant blue (CBB) staining. The purified protein
concentration was determined using a NanoDrop spectrophotometer, measuring the
absorbance A280. The final yield of purified MTH1 was approximately 0.65 g/l of culture
(Figure 15B). To remove imidazole, the purified protein was dialyzed using 1X-PBS and
concentrated in concentrator columns (5 kDa cutoff range).
CD Spectroscopy Ascertaining the Folding Conformation of MTH1
To confirm the folding of the purified MTH1 protein, circular dichroïsm (CD)
spectrophotometric analysis was performed. The CD spectra of MTH1 was studied and the
secondary structure was monitored. The signals recorded between 190 and 260 nm were
superposed. Three consecutive spectral scans were averaged, the corresponding blank buffer
was subtracted, and the data was expressed as the mean residue molar ellipticity, MRME (deg
cm2/dmol). The secondary structure calculation was performed using K2D3 software
(http://cbdm-01.zdv.uni-mainz.de/~andrade/k2d3/) for spectrum analysis to quantify the α
helix, β sheets, and random coil content [47]. The far-UV spectra of the proteins displayed an
approximate 3.67% helical character and 35.98% beta sheet against an expected structure of
14% helical and 40% beta sheet (Figure 16).
Bio-Physical Evaluation of Emodin Binding MTH-1 for Functional Inhibition
Presently, surface plasmon resonance (SPR) is one of the most widely used techniques for the
biophysical assessment of small molecule interactions to develop target‐based drugs. The
SPR assay was performed to assess the real time binding of MTH1 with emodin at
physiologically relevant concentrations in real time, establishing their affinity. Being a
surface technique, it allows for the performing of experiments at a much lower concentration
range. Using MTH1 protein as a ligand (covalently coated to an activated monolayer of self-
assembled 11-mercaptoundecanoic acid [MUA] on a gold surface, following the amine
coupling method) and different concentrations of emodin and (S)-crizotinib as analytes, SPR
was performed. The kinetic analysis sensorgrams (Figure 17A) showed a concentration
dependent increase in the binding of emodin and (S)-crizotinib during the association phase.
The binding affinity was found to be significant with a calculated KD value of 22.2 ± 0.68
µM for emodin and 4.04 ± 0.43 µM for (S)-crizotinib (positive control), ascertaining that
emodin has a role in MTH1 inhibition as its highly probable interacting partner (Figure 17B).
8-OHdG quantification to assess DNA damage due to Emodin mediated MTH1 functional
inhibition
These observations that the DNA damage occurring due to emodin mediated MTH1
functional inhibition were substantiated by a considerable increase in the 8-OH deoxy-
guanosine levels in the DNA from NSCLC cells, quantified by using ELISA. Emodin
treatment resulted in a dose and time dependent increase in the concentration of 8-hydroxy-
2’-deoxyguanosine (nM)/ DNA (μM/ml) in all the three NSCLC cell lines (Figure 18). In A-
549 the concentration of 8-OH-dG showed an increase from approximately 130 nM to 185
nM from 24 to 72 h. Likewise in NCI-H-460, the concentration of 8-OH-dG showed an
increase from approximately 160 nM to 200 nM from 24 to 72 h. In NCI-H-520 the
concentration of 8-OH-dG showed an increase from approximately 140 nM to 175 nM from
24 to 72 h. Taken together, emodin treatment thus induces MTH1 functional inhibition and
augments the concentration of oxidized dNTPs, enhancing DNA oxidative damage and
subsequent cancer cell death.
FIGURES
A B
Figure 1: A: Stereo view of the MTH1 binding site is shown. The protein portions building up the binding site are colored as
pink mesh and emodin as ligand (Green) is embedded deep in the active site of MTH1. B: Molecular interactions between
MTH1 protein and emodin, showing the hydrophobic interactions (in red) and hydrogen bonds (in green). The diagram is
illustrated using LIGPLOT software
A B
Figure 2: Emodin-MTH1 complex - Graphical representation of A: The RMSD analysis of the 50 ns trajectory to examine
the detailed dynamics of all binding atoms. B: The RMSF analysis. C: The radius of gyration (Rg) analysis. D: The SASA
analysis. E: The H-bond lengths of the simulated complex.
A
B
C
Figure 3: Measurement of cell proliferation by Trypan Blue assay. A, B and C are graphical representation of the Total, Live
and Percent Dead cells caused by increasing emodin treatment (0, 1, 10, 25, 40, 50 and 75 μM) at 24, 48 and 72 h, as
measured by phase-contrast for NCI-H-460 (Large cell carcinoma), A-549 (adenocarcinoma) and NCI-H-520 (squamous cell
carcinoma) cell lines respectively. Data shown as mean ± S.E.M. of three independent experiments and * indicate P values <
0.05.
A B
C
Figure 4: Measurement of cell proliferation by MTT assay. A, B and C are graphical representation of percent inhibition
caused by increasing emodin treatment (0, 10, 25, 50, 75, 100, 150 and 200 μM) at 24, 48 and 72 h, for NCI-H-520
(squamous cell carcinoma), NCI-H-460 (Large cell carcinoma) and A-549 (adenocarcinoma) cell lines respectively as
measured by MTT colorimetric assay, absorbance taken at 595nm. IC50 values are calculated from sigmoidal fitting of
growth curves. Data shown as mean ± S.E.M. of three independent experiments.
A
B
C
D
E
F
Figure 5: Cell cycle analysis. A, B and C are the graphical representation of the number of cells in G1, S and G2/M phases
of NSCLC cells with emodin treatment (0, 25, 50 and 75 μM) at 24, 48 and 72 h, as measured by BD FACS Aria Flow
Cytometer for NCI-H-520 (squamous cell carcinoma), A-549 (adenocarcinoma) and NCI-H-460 (Large cell carcinoma) cell
lines respectively. D: Flow cytometric plots of NCI-H-520 cells with emodin treatment (0, 25, 50 and 75 μM) at 24, 48 and
72 h. Western Blot Analysis of Cell Cycle proteins. E: Protein level expression profile of CDK-4, Cyclin B1, CDK-2 and
Cyclin D1 respectively. F(a-d): The graphical representation of the protein levels quantified by Image-J software. The data
are representative of three independent experiments carried out under the same conditions.
A
B
C
D
E
F
G H
I
Figure 6: Measurement of Apoptosis by Annexin V- PI assay. A, B and C are Representative plots obtained from flow
cytometry for NCI-H-460 (Large cell carcinoma), A-549 (adenocarcinoma) and NCI-H-520 (squamous cell carcinoma) cells
under increasing emodin treatment (0, 25, 50 and 75 μM) at 24, 48 and 72 h respectively. The lower left quadrant indicates
the viable cells. The lower right quadrant represents the annexin V positive/propidium iodide (PI) negative staining
indicating early apoptotic cells. The upper right quadrant represents high annexin V and PI staining both, indicating late
apoptosis and the upper left quadrant represents low annexin V and high PI staining indicating necrosis. D, E and F are the
graphical representation of percent inhibition for NCI-H-520 (squamous cell carcinoma), A-549 (adenocarcinoma) and NCI-
H-460 (Large cell carcinoma) cells respectively. G and H: Protein level expression profiles of Survivin, Bax, Bcl-2 and
PARP and Caspase-3 cleavage respectively. I: The graphical representation of the protein levels quantified by Image-J
software. Data shown as mean ± S.E.M. of three independent experiments.
A
B
C
D
E
F
G
H
Figure 7: Wound scratch assay. A, B and C are photographical representation of the visual wound healing under increasing
emodin treatment (0, 25, 50 and 75 μM) at 24, 48 and 72 h, as measured by phase-contrast for NCI-H-520 (squamous cell
carcinoma), A-549 (adenocarcinoma) and NCI-H-460 (Large cell carcinoma) cell lines respectively. D, E and F are the
graphical representation of scratch areas for A, B and C respectively. G: Protein level expression profiles of Vimentin and
Integrin β1 respectively. H: The graphical representation of the protein levels quantified by Image-J software. Data shown as
mean ± S.E.M. of three independent experiments.
A B
C D
Figure 8: A: Confocal microscopic images of NCI-H-520 cell line stained with CellROX™ Deep Red, showing increasing
red fluorescence (increasing ROS). B: Confocal microscopic images of NCI-H-520 cell line stained with CellROX™ Green,
showing increasing green fluorescence (increasing ROS). C and D: Graphical representation of the mean fluorescent
intensity/ nuclei indicating an increase in the fluorescence signal of ROS in cells with increasing emodin treatment for 24h,
as observed under the confocal microscope for A and B respectively. Cells of interest have been highlighted with arrows in
RED.
A
B
C D E
Figure 9: Mitochondrial membrane potential analysis by JC-1 staining. A: Confocal microscopy images of NCI-H-520
(squamous cell carcinoma) cells with emodin treatment (0, 10, 25, 50 and 75 μM) and CCCP (positive control) at 24h,
stained with JC-1 dye. B : graphical representation of the fluorescent intensities depicted as the ratio of monomer:aggregates
from the Confocal microscopy images of NCI-H-520 (squamous cell carcinoma) cells represented in A. C, D and E shows
the graphical representation plots of fluorescent intensities (ratio of monomer:aggregates) obtained from fluorimetry for
NCI-H-520 (squamous cell carcinoma), A-549 (adenocarcinoma) and NCI-H-460 (Large cell carcinoma) cells under
increasing emodin treatment (0, 10, 25, 50, 75, 100, 150 and 200 μM) at 24, 48 and 72 h respectively. The data shown as
mean ± S.E.M. of three independent experiments carried out under the same conditions. Cells of interest have been
highlighted with arrows in RED.
A
B
C D E
Figure 10: Mitochondrial membrane potential analysis by DIOC6 staining. A: Confocal microscopy images of NCI-H-520
(squamous cell carcinoma) cells with emodin treatment (0, 10, 25, 50 and 75 μM) and CCCP (positive control) at 24h,
stained with DIOC6 dye. B: graphical representation of the fluorescent intensities of the Confocal microscopic images of
NCI-H-520 (squamous cell carcinoma) cells represented in A. C, D and E shows the graphical representation plots of
fluorescent intensities obtained from fluorimetry for NCI-H-520 (squamous cell carcinoma), A-549 (adenocarcinoma) and
NCI-H-460 (Large cell carcinoma) cells under increasing emodin treatment (0, 10, 25, 50, 75, 100, 150 and 200 μM) at 24,
48 and 72 h respectively. The data shown as mean ± S.E.M. of three independent experiments carried out under the same
conditions. Cells of interest have been highlighted with arrows in RED.
A
B
C
Figure 11: Analysis of DNA fragmentation induced by emodin treatment. A, B and C are photographical representation of
the visual ladder like DNA bands formed under increasing emodin treatment (0, 25, 50 and 75 μM) at 24, 48 and 72 h, as
assessed by 1% agarose gel electrophoresis and ethidium bromide staining for NCI-H-520, A-549 and NCI-H-460 cell lines
respectively. L: Ladder, C: Control, 25: 25 µM emodin, 50: 50 µM emodin, 75: 75 µM emodin. The data are representative
of three independent experiments carried out under the same conditions. Gel bands of interest have been highlighted with
arrows in RED.
A
24 hr (10X)
Control 25µM 50 µM 75 µM
24 hr (60 X)
Control 25µM 50 µM 75 µM
B
48 hr (10X)
Control 25µM 50 µM 75 µM
48 hr (60 X)
Control 25µM 50 µM 75 µM
C
D E
F
72 hr (10X)
Control 25µM 50 µM 75 µM
72 hr (60 X)
Control 25µM 50 µM 75 µM
Figure 12: Analysis of Comet Assay. A, B and C are photographical representation of the visual comet like DNA from cells
formed under increasing emodin treatment (0, 25, 50 and 75 μM) at 24, 48 and 72 h (at 10X and 60X), as assessed by single
cell gel electrophoresis and ethidium bromide staining, observed under confocal microscope for NCI-H-520 (squamous cell
carcinoma). D, E and F are graphical representations of the comet tail moments of NCI-H-460 (Large cell carcinoma), A-
549 (adenocarcinoma) and NCI-H-520 (squamous cell carcinoma) cell lines respectively. Data shown as mean ± S.E.M. of
three independent experiments and * indicate P values < 0.05. Cells of interest have been highlighted with arrows in
GREEN.
A
B
C
D
Figure 13: Analysis of TUNEL Assay. A, B and C are the Confocal microscopy images of NCI-H-520 (squamous cell
carcinoma), A-549 (adenocarcinoma) and NCI-H-460 (Large cell carcinoma) cells under emodin treatment (50 μM) at 24, 48
and 72 h along with vehicle controls respectively stained with the TUNEL assay protocol. Increasing Green fluorescence
indicates increasing DNA Damage. D: graphical representation plot of fluorescent intensities obtained from Confocal
microscopy images for A-C. The data shown as mean ± S.E.M. of three independent experiments carried out under the same
conditions. Cells of interest have been highlighted with arrows in RED.
A
B
C
D
Figure 14: Analysis of DNA damage and repair activation. A, B and C are the Confocal microscopy images of NCI-H-520
(squamous cell carcinoma), A-549 (adenocarcinoma) and NCI-H-460 (Large cell carcinoma) cells under emodin treatment
(50 μM) at 24h along with vehicle controls respectively stained with ATM-p, DNA-PKCs and 53 BP-1 Primary antibodies
subsequently stained with Alexa-488 labelled Secondary antibodies. Increasing Green fluorescence indicates increasing
expression of the DNA Damage and repair induction specific proteins. D: Protein level expression profiles of ATM-p, DNA-
PKCs and 53 BP-1 respectively. The data shown is representative of three independent experiments carried out under the
same conditions. Cells of interest have been highlighted with arrows in RED.
Figure 15: A: Codon Plus protein expression study on SDS page gel where Lane 1: un-induced @37°C for 1 h, Lane 2: IPTG
induced @37°C for 1 h, Lane 3: un-induced @37°C for 3 h, Lane 4: IPTG induced @37°C for 3 h, Lane 5: un-induced
@18°C overnight and Lane 6: IPTG induced @18°C overnight. B: Codon Plus protein purification study on SDS page gel
where Lane 1: Cell Pellet, Lane 2: Before Sonication, Lane 3: Total Cell Lysate, Lane 4: Lysate supernatant, Lane 5: Lysate
Flow Through, Lane 6: 10 mM Imidazole Wash, Lane 7: 25 mM Imidazole Wash, Lane 8: 150 mM Imidazole Elution 1,
Lane 9: 150 mM Imidazole Elution 2, Lane 10: 150 mM Imidazole Elution 3, Lane 11: 250 mM Imidazole Elution 1, Lane
12: 250 mM Imidazole Elution 2, Lane 13: 250 mM Imidazole Elution 3, Lane 14: 250 mM column wash. The data shown is
representative of three independent experiments carried out under the same conditions.
Figure 16: Secondary structure analysis by circular dichroism. Spectra of the MTH1 protein is presented in molar ellipticity
(deg.cm2.dmol-1). The spectrum was recorded at 25 °C in a 0.01 cm path cell length quartz cell using MTH1 protein at a
concentration of 5 μM in a 10 mM Tris-HCl buffer (pH 7.4). Red: input structure; Green: predicted structure. An average of
three consecutive spectral scans was used and corresponding blank buffer was subtracted.
A
B
Figure 17: Surface Plasmon Resonance sensorgram. A: SPR sensorgram showing surface competition assay of MTH1
protein (ligand) with emodin and (S)-crizotinib (analytes). As the concentration of the analyte molecule increased, the SPR
response increased. B: plot of the mean Kd values of MTH1 protein (ligand) with emodin and (S)-crizotinib (analytes). An
average of three Kd values was taken for plotting the mean.
Figure 18: Quantification of 8-hydroxy-2’-deoxyguanosine (nM)/ DNA (μM/ml). A-549 (adenocarcinoma), NCI-H-460
(Large cell carcinoma) and NCI-H-520 (squamous cell carcinoma) cells under emodin treatment (25, 50 μM) at 24, 48 and
72 h along with vehicle controls respectively as quantified using 8-OH-dG ELISA protocol.
TABLES
Table 1: Measurement of the Binding affinity scores, energies, molecular interactions as well as H-bond distances of the
highest scoring docked compounds with MTH1 using XP docking protocol.
Compound
Glide score
(XP)
(kcal/mol)
Interacting residues Distance (H-bond,
Å)
Glide
energy
(kcal/mol)
Ligand_71308639
(Taxifolin
hydrate)
(Sigma
Phytochemical
compounds
library)
-8.76
Gly28, Asn33, Asp119, Thr8,
Lys23, Phe27, Arg31, Asn33,
Gly 34, Phe72, Met81,
Trp117
2.95
3.28
3.30
-43.26
Ligand_148
(Quercetin
dihydrate)
(Natural remedies
compound
Library)
-8.74
Asn33,
Gly36,
Leu9, lys23, Phe27, Gly 34,
Phe35,Glu56, Phe72, Met81,
Trp117, Asp119,
Asp120,Phe139
3.21
3.01
-45.37
Ligand_3220
(emodin)
(Sigma
Phytochemical
compounds
library)
-7.63
Gly36,
The8, Leu9, lys23, Phe 27,
Gly 28, Asn33, Gly37, Ile70,
Phe72, Val83, Trp117,
Asp119, Try123
3.22
-29.43
Compound emodin Taxifoline hydrate Quercetine Dihydrate
Docking
Score -6.83 -5.77 -5.81
Table 2: Measurement of the Docking scores of lead compounds with MTH1 using AutoDock 4.0.1 software protocol.
MMPBSA (Kcal) MM (Kcal) PB (Kcal) SA (Kcal) Vdw (Kcal) Elec (Kcal)
-167.8995024 -425.5168452 271.5267 -13.9094 -168.68 -256.837
Table 3: Measurement of the Molecular mechanics energy in combination with the generalized Born or Poisson–Boltzmann
and surface area solvation (MMPBSA) parameters.
Discussion
Interestingly, the MTH1 protein is essential to efficient survival in cancer cells but its
function is not crucial for normal cells, creating research interest in developing anti-cancer
strategies based on MTH1 inhibition. Cancer cells are marked by dysfunctional redox
regulation. This accumulation of ROS creates a cellular environment of oxidized nucleotides
that make it essential for the MTH1 protein function to prevent their mis-incorporation and
subsequent DNA damage during augmented replication and rapid division. MTH1 is
overexpressed in several cancers and its catalytic function is noticeably augmented in lung
cancers as a non-oncogene addiction function and a response to survive by circumventing the
nuclear incorporation of damaged dNTPs [22]. Since current anti-cancer approaches are
dominated by target proteins essential to normal cells, causing severe side effects when
treated with chemotherapies, MTH1 inhibition is a promising anti-cancer approach. The
current work aims to identify novel natural small molecular inhibitors to the Nudix hydrolase
protein (MTH1) to be used as an anti-cancer therapeutic.
Natural compound libraries were screened to obtain MTH1 active site binding moieties and
validated the most promising, emodin, for its effect in NSCLC cell line models upon MTH1
inhibition. It was validated that emodin treatment on NSCLC caused a surge in ROS levels,
loss of mitochondrial potential, genome-wide double-stranded DNA damage, and apoptosis.
Emodin caused a dose and duration dependent decline in the proliferation of NSCLC cells, a
G2/M phase cell cycle arrest, decrease in the cyclin-D1, CDK-2, and CDK-4 and an increase
in cyclin-B1 protein levels. There was also a rise in the number of apoptotic cells, protein
level expression profiles of Bax (pro-apoptotic), survivin and decrease in Bcl-2 (anti-
apoptotic) in a duration dependent manner. The cleavage of caspase-3 and PARP increased,
as did the p-21 levels. Emodin caused an increase in mitochondrial and cytoplasmic ROS
levels and significantly reduced the mitochondrial membrane potential. Emodin caused
extensive DNA damage and fragmentation, with an increase in the repair protein levels
ATM-p, DNA-PKCs, and 53 BP-1. We observed the mechanism of emodin action:
augmented ROS levels causing extensive dNTP oxidation, subsequent DNA damage, and
apoptosis via MTH1 inhibition. Since the DNA damaging effects of emodin treatment were
reported earlier [29, 37] but its mechanism of action had not been elucidated clearly, this
paper highlights for the first time that the effect of emodin is via the mechanism of MTH1
inhibition. We expressed the human MTH1 protein in an E. coli bacterial system to perform
binding analysis of emodin with MTH1 protein using SPR studies. Emodin binds MTH1 in a
concentration dependent manner, proving the genotoxic and cytotoxic effects of emodin are
due to its capability to inhibit MTH1 function in cancer cells.
The effect of emodin highlights a novel concept to exploit the unique oxidative status of
tumors, increase ROS, and concurrently target the redox-protective MTH1 function for
cancer therapy. Thus, the mode of emodin targeting the nucleotide pool homeostasis is a
promising cancer cell specific natural small molecule therapeutics.
Conclusion
The tumor specific unique redox status makes the rapidly proliferating cells more dependent
on redox protective mechanisms such as MTH1 upregulation, opening a novel therapeutic
window for the selective induction of cancer cell apoptosis via oxidative stress overload,
sparing the normal cells. The dual dNTP pool targeting of emodin (increasing ROS on one
hand and inhibiting oxidized dNTP pool sanitation by MTH1 on other) shows an additive
effect on apoptotic cancer cell death, highlighted by augmented DNA damage and
corresponding ATM repair pathway upregulation. Conclusively, we demonstrate that MTH1
protein targeting using a novel natural small molecular inhibitor, emodin, constitutes a simple
and safe anti-cancer therapeutic approach that aims to enhance the deregulated redox
metabolism and increase ROS levels in cancer to cause extensive DNA damage specific to
cancer cells and therefore leading to senescence.
Acknowledgements
The authors thank Jawaharlal Nehru University (JNU) for the infrastructural support. DW
and DS acknowledge scholarship support from DST-INSPIRE (Grant No. DST/INSPIRE
Fellowship/2014/228) and DST-SERB (Grant No. PDF/2016/000245) respectively. Authors
also acknowledge the Advanced Instrumentation facility, AIRF-JNU for the Surface Plasmon
Resonance (SPR) Spectrometer - Autolab ESPRIT and Nikon Real Time Laser Scanning
Confocal Microscope Model 80i. The authors also acknowledge the fellow lab-mates Nidhi
Shrivastava for aiding in MTH1 protein purification, Aditi Singh for performing MD
simulation and Anchala Kumari for performing Autodock.
Conflict of interest
The authors declare that they have no conflicts of interest with the contents of this article.
Author Contribution
DW designed, performed and analysed the experiments and wrote the paper. DS performed
comet assay and aided in performing experiments and proofreading of the paper. AG
conceived and supervised the study and manuscript.
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Supplementary Materials and Methods
Screening of MTH-1 inhibitors
Protein preparation
The native human MutT Homolog 1 (MTH1) protein X-ray crystallography resolved
structure, having 8-oxo-dGTPase function (Nudix hydrolase family) (PDB ID: 3Q93), with a
resolution of 1.8 Å was retrieved for the study from the RCSB protein repository known as
the Protein Data Bank (PDB) [1]. The PDB structure contained 154 amino acids from 3-156
residues along with water. For all subsequent analysis, the water molecules contained in the
structure, were removed using the Accelrys Viewerlite 5.0 software [2]. The retrieved protein
structure was next pre-processed, adjusted and optimized for further analysis with the aid of
the inbuilt Protein Preparation Wizard embedded in software Schrodinger Suite, Maestro
interface for docking studies [3]. The 3D structure processing involved the addition and
optimization of hydrogen bonds by fixing bond orders, bond lengths, removal of bad contacts
and steric clashes, creation of disulfide bonds and stabilizing ionization states, protein
terminal capping, conversion of the seleno-methionine residues to methionine and fixing of
the missing amino acids and side-chain orientations. The prepared MTH1 structure was
further optimized to acquire an energetically minimized conformation using the OPLS
(Optimized Potentials for Liquid Simulations) force field [4]. Further flexible filtering was
applied for discarding the ligands with poor pharmacological characteristics by applying the
Lipinski’s filter [5].
Ligand preparation
For the screening purposes, we created a virtually available library of ligands by extracting
the natural compounds from the freely available special subsets of two libraries, first, the
phytochemical compound library from compounds available at Sigma and Natural Remedies.
For the docking approach, the libraries were prepared and processed using the Schrodinger
Suite, LigPrep Wizard [6]. For inclusion of all ligand conformations, all the possible
stereochemical, tautomeric or ionization variants of the ligand molecules were created for
screening and subsequent energy minimization.
Grid generation
With the aid of the Receptor Grid generation panel available in the Glide docking module,
Schrodinger suite, a grid using the centroid of the following residues: Leu9, Phe27, Asn33,
Phe72, Met81, Val83, Trp117, Asp119, Asp120, Trp123 and Phe139, was generated with a
radius of 20Å. The parameters such as the scaling factor was kept at 1.0, the partial charge
cut-off was set at 0.25 (pre-set values) and no additional constraint values were used [7-10].
Virtual screening: The high throughput virtual screening (HTVS) and extra precision (XP)
docking
The growing need of efficient and rapid search for novel binding partners to proteins, has
paved the way for the emergence of Virtual screening as an extremely effective filtering tool
for the process of computer aided drug-designing. It has been extensively applied for
computing of the binding affinities and elucidation of the binding modes for numerous ligand
receptor complexes.
For lead ligand identification, the prepared ligand library was docked into the active site of
the MTH1 protein employing the Schrodinger’s Glide module also known as the Grid-based
Ligand Docking along-with its Energetics [7, 9]. The Glide algorithm is derived from a
systematic virtual screening protocol using the searching algorithm based on incremental
construction for creating an output called as the G-Score scoring function, combined with a
series of parameters [11]. Computationally, Glide performs a ligand specific extensive search
for the conformation, location and orientation binding parameters over the protein active site
to generate an output in terms of the Glide Score (GScore) and Glide energy.
A two-step docking strategy for performing the virtual screening was employed: The high-
throughput virtual screening (HTVS) and the extra precision (XP) docking employing the
Glide virtual screening workflow (GVSW) [7]. The essential grid coordinates were first
employed to perform the MTH1 inhibitor screening using the HTVS docking algorithm of
Glide [9]. A rational virtual screening workflow from HTVS to XP is performed using Glide,
obtaining enriched data at each level to shortlist fewer compounds for studying higher
precision levels. A docking Score of -7kcal/mol threshold was set for compound screening
and for obtaining higher precision docking, the compounds above the threshold values were
subsequently subjected to the extra precision Glide (XP), that is a higher accuracy docking
algorithm that ensues more advanced screening [11]. The three natural compounds with top
docking scores with MTH1were selected and their results are tabulated in Table 1.
Validation of docking by AutoDock
The docking results from the Glide module were subjected to further confirmation using the
AutoDock software Molecular Docking Suite version 4.0, using the same crystal structure as
above [12]. The missing atoms were repaired, heteroatoms were removed, polar hydrogens
were added for ionization correction and amino acid residue tautomeric states and the non-
polar hydrogens were merged from the protein for its preparation for Autodock. For the
generation of energy based AutoDock scoring function, hydrogen bonding, entropy losses,
electrostatic and van der Waals forces were studied [12, 13]. The parameters of Gasteiger
charges and rigid roots were allocated to the lead ligands and scoring grid of the energy
function of 60 Å × 60 Å × 60 Å (x, y, z) dimensions was generated for all the key residue
incorporation. The adaptive local searching protocol of the default parameters of the
Lamarckian genetic algorithm were used to identify a maximum fitting conformation.
Molecular dynamics
Molecular dynamics simulations of docked complex
The structure of MTH1 protein bound to the lead compounds was analyzed using the Ligplot
program for hydrogen bonding and other non-bonded interaction calculation between the
atoms of ligand and protein [14]. Also, the three-dimensional structural models of the
complex of MTH1 protein bound to the lead compounds were viewed in the PyMOL program
to study how well the compounds were fitting inside the protein active site.
We further employed Molecular dynamics (MD) simulation as a protocol to perform further
stability study of the lead compound-MTH1 binding. The native MTH1 protein was used for
docking with the lead compound, emodin and the energy minimized stable structure was used
for performing MD simulation studies for 50 ns time. The simulation was run using the
GROMACS software version 5.0, employing the GROMOS96 43a1 force field in a cubic box
with water model spc [15, 16]. For the Gromos force field, iGROMACS compatible ligand
topologies were created with the help of the PRODRG server [17]. The system underwent
energy minimization up till a maxima of <10.0 kJ/mol/nm was achieved at 50,000 ns steps,
using the steepest descent algorithm. The LINCS algorithm was used for bond lengths
constraining [18]. The unfavorable contacts between molecules were relaxed using the Verlet
cut-off scheme. Molecular dynamics was run on the equilibrated system for 50 ns along with
time-step integration of 2 fs employing the leap-frog integrator. The MD trajectory frames
were saved for every 10 ps. The cluster analysis approach representative protein frame
achieved from the 50 ns trajectory was quality assessed for geometrical and energy
parameters. The trajectory files were analyzed using numerous statistical parameters of the
various inbuilt scripts of GROMACS.
RMSD and RMSF Analysis
The RMSD (root mean square deviation) analysis is used to understand the ligand-protein
complex stability and simulation convergence time. The RMSF (root mean square
fluctuation) value noticeably illustrates the protein segment residue flexibility differences that
correlate to the numerous intermolecular hydrogen bonding interactions and contacts of
hydrophobic nature identified along the analysis. The RMSD and RMSF values of the
MTH1-emodin complex were calculated taking reference of the first frame along the whole
simulation trajectory.
Radius of gyration
The atomic distribution from the center of mass that is common to all molecules, calculated
as the mass-weighted root mean square distance (RMSDmw) in the system is called as the
radius of gyration. Any protein mutation can cause an increase or decrease in protein Rg as
each protein comprises of its own characteristic radius of gyration. The Radius of gyration is
also affected after favorable ligand binding as it determines the protein compactness and
overall protein complex dimensions, responsible for their respective interactions. The radius
of gyration for emodin-MTH1 complex was computed throughout the 50 ns trajectory.
H-Bond Length analysis
Hydrogen bonds also play a crucial role in the protein structure stability. The protein-drug
complex intermolecular H-bonding pattern along with other interactions was studied for 50 ns
simulation and analyzed describing the complex stability. The MD simulations of 50 ns
reveal significant outcomes reflecting the structural stability, protein dynamics and
interactions for the binding mechanism elucidation.
MMPBSA binding free energy calculation
MMGBSA and MMPBSA (Molecular mechanics energy with generalized Born or Poisson–
Boltzmann and surface area solvation) methodology is regularly being implied estimation of
the biological macromolecule and small ligand free interaction energies [19]. Considering the
free and interacting state free energy differences of the two moieties to provide comparative
free energies for them in distinct solvated conformations, comprises the main objectives of
these approaches. The binding free energies for the Protein drug interaction were computed
using the MMPBSA methodology, to create accurate calculations of the drug binding affinity
relative to its target proteins.
The g_mmpbsa package was used to calculate the binding free energy of emodin, employing
the MMPPSA method. Here, the ∆Gbinding is computed from free energies of the receptor
ligand complex with the equation:
∆Gbinding = Gcomplex - Gligand - Greceptor
For the emodin- MTH1 complex, a 50 snapshot trajectory file was used in a 200 frame time-
step for estimation of the free binding energy, electrostatic energy, Van der Waals (VDW),
polar solvation and the solvent-accessible surface area (SASA) energy.
Assessment of cell morphology
NSCLC cells were cultured until 80% confluence, then seeded in 24 well plates in 500 μL of
RPMI medium at a cell density of approximately 105 cells/well. The cells were treated with
emodin at different dilutions (0, 25, 50 and 75 μM) in dimethyl sulfoxide (DMSO), DMSO
vehicle < 0.5% volume) for 24, 48 and 72 h. Then, post treatment the cells were examined
under a phase-contrast microscope (Zeiss Primovert) and photographed.
Assay of cell growth and Viability: IC50 determination
Trypan Blue dye exclusion Assay:
NSCLC cells were cultured until 80% confluence, then seeded in 24 well plates in 500 μL of
RPMI medium at a cell density of approximately 105 cells/well. The cells were treated with
emodin at different dilutions (0, 1, 10, 25, 40, 50 and 75 μM) in DMSO (vehicle < 0.5%
volume) for 24, 48 and 72 h. Then, post treatment the cells were centrifuged, washed with
PBS and then suspended in PBS (pH 7.4) containing 0.4% trypan blue solution.
The percent live and dead cells was determined using a haemocytometer for counting the
cells stained with trypan blue. The cells with a properly defined cellular outline, excluded the
dye and were scored as alive. Whereas the cells not excluding it were scored as dead. The
total cell number, live cell number and percentage of dead cells was calculated from three
independent experiments.
Total cell count = Total Live cell count + Total Dead cell count
% Dead cells = (Total Dead cell count/ Total cell count) * 100
MTT Assay:
NSCLC cells were cultured until 80% confluence, then seeded in 96-well plates in 100 μL of
RPMI medium at a cell density 104 cells/well. The cells were then treated with emodin at
different dilutions (0, 10, 25, 50, 75, 100, 150 and 200 μM) in DMSO (vehicle < 0.5%
volume) for 24, 48 and 72 h.
After incubation, the treatment containing media was removed and 100 μL of working 3-(4,
5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) stock solution (0.5 mg/mL
prepared culture media) was added to each well and the cells were incubated for 3 h in a
humidified atmosphere containing 5% CO2. The MTT containing media was carefully
aspirated, without disturbing the adherent cells and the formazan that formed was solubilized
from the cells by adding 100 μL of DMSO to each well. The colour intensity of formazan
was measured by the absorbance at 595 nm with a 96-well ELISA plate spectrophotometer
(2300 EnSpire Multimode Plate Reader, PerkinElmer). The rate of tetrazolium reduction to
formazan is directly proportional to the rate of cell proliferation and subsequent cell viability.
MTT assays were performed in triplicate and each measurement contained three parallels
(n=9).
% inhibition = 100 – 100 × (A595nm) treated/ (A595nm) control
% cell viability = 100 - % inhibition
Wound- healing Assay
NSCLC cells were cultured until 80% confluence, then seeded in 6 well plates in 3ml of
RPMI medium at a cell density of approximately 105 cells/ml and grown till the formation of
a confluent monolayer. The confluent cell monolayer was scraped using a p200 pipet tip in
a straight line to create a “scratch/wound”. The cells were washed to remove debris with 1
ml RPMI and fresh 3ml RPMI media was added for the in vitro scratch/wound assay. The
cells were subsequently treated with emodin at different dilutions (0, 25, 50 and 75 μM) in
DMSO (vehicle < 0.5% volume) for 24, 48 and 72 h. Then, post treatment the cells were
washed (2 times) with PBS and observed under phase-contrast microscope (Zeiss Primovert)
and photographed. The images were analysed using ImageJ software (Wound Healing Tool).
Mitochondrial membrane potential (ΔΨM) assay
JC-1 and DIOC6 [3] iodide dyes: Microscopic Assessment of Mitochondrial membrane
potential
NSCLC cells were cultured until 80% confluence, then seeded on coverslips in 24 well plates
in 500 μL of RPMI medium at a cell density of approximately 105 cells/well. Then they were
treated with emodin at different dilutions ( 0, 1, 10, 25, 40, 50 and 75 μM ) in DMSO (vehicle
< 0.5% volume) for 24, 48 and 72 h. Carbonyl cyanide m-chlorophenyl hydrazone (CCCP),
that functions as a mitochondrial uncoupler and a chemical inhibitor of oxidative
phosphorylation causing mitochondrial depolarization, was used as a positive control at a
concentration of 50 µM, for 10 min. Post- treatment, the media above the coverslips was
aspirated and 300 µl of fresh media containing - 1µg/ml JC-1 (working concentration from
1mg/ml JC-1 in DMSO stock solution) or 1µM DIOC6 [3] iodide (working concentration
from 1mM DIOC6 [3] iodide in DMSO stock solution) added to each well. Then cells on
coverslips were incubated at 37°C in humidified 5% CO2 chamber for 20 min. Post
incubation they were washed (2 times) with PBS (pH 7.4) and stained with a 2µg/ml working
Hoechst staining solution from a Hoechst 10 mg/mL stock in PBS for 10 min at room
temperature, in dark. The cells were washed (2 times) with PBS and the coverslips were
mounted in 1,4-diazabicyclo [2.2.2] octane (DABCO) (Sigma Aldrich) Mounting Medium
(containing 1% DABCO (w/v) in 80% glycerol and 20% PBS). The coverslips were acquired
immediately and analyzed with a Nikon Real Time Laser Scanning Confocal Microscope at
60X magnification with supporting software.
Nuclear morphological Assessment (Hoechst 33342 staining)
NSCLC cells were cultured until 80% confluence, then seeded on coverslips in 24 well plates
in 500 μL of RPMI medium at a cell density of approximately 105 cells/well. The cells were
treated with emodin at different dilutions (0, 25, 50 and 75 μM) in DMSO (vehicle < 0.5%
volume) for 24, 48 and 72 h. Post treatment the cells were washed (2 times) with PBS and
stained with a 2µg/ml working Hoechst staining solution from a Hoechst 10 mg/mL stock in
PBS for 10 min at room temperature, in dark. The cells were washed (2 times) with PBS and
fixed with 4% paraformaldehyde solution for 15 min at room temperature. Post fixing, the
cells were washed (2 times) with PBS and the coverslips were mounted on DABCO (Sigma
Aldrich) Mounting Medium (containing 1% DABCO (w/v) in 80% glycerol and 20% PBS).
The Images were acquired and analysed with a Nikon Real Time Laser Scanning Confocal
Microscope at 60X magnification with supporting software.
Anenexin V/PI Apoptosis Analysis
Microscopic Assessment of Apoptosis
For confocal microscopy, NSCLC cells were cultured until 80% confluence and seeded on
coverslips, placed in 24-well plates at a cell density of approximately 104 cells/well and the
cells were treated with emodin at different dilutions (0, 25, 50 and 75 μM) in DMSO (vehicle
< 0.5% volume) for 24, 48, 72 h; following which the cells were washed (2 times) with PBS.
The cells on coverslips were then stained with 5 µL Alexa Fluor® 488 annexin V and 1 µL
100 µg/mL PI working solution (PI) per 100 µL in each well, in dark at room temperature for
15 min, according to the manufacturer’s instruction (Dead Cell Apoptosis Kit with Annexin
V Alexa Fluor™ 488 & Propidium Iodide (PI), Thermo Fisher Scientific). After a PBS wash,
the DNA was counterstained with a 2µg/ml working Hoechst staining solution from a
Hoechst 10 mg/mL stock in PBS for 10 min at room temperature and the coverslips were
mounted in DABCO (Sigma Aldrich) Mounting Medium (containing 1% DABCO (w/v) in
80% glycerol and 20% PBS). Immediately, images were acquired using fluorescence
emission at 530 nm and >575 nm and analysed with a Nikon Real Time Laser Scanning
Confocal Microscope at 60X magnification with supporting software.
Cell lysate Preparation
NSCLC cells were grown in T25 flasks to 60% confluence. Then after 18 h, the cells were
treated with emodin at 50 μM dilution in DMSO (vehicle < 0.5% volume) or vehicle alone
for 24, 48, 72 h. After treatment both the floating and adherent cells were collected and
washed twice with PBS. The cells were then pelleted down at 1000 rpm for 3 min. 100µl of
lysis buffer (25mM Tris, pH 7.4, 150 mM NaCl, 0.1% SDS, 1% Nonidet P-40, Protease
inhibitor cocktail (Sigma Aldrich)) was added to each sample and the pellet was resuspended.
The samples mixed with lysis buffer were kept on ice for 30 minutes with gentle intermittent
tapping. The after which the samples were pelleted at 15000 rpm for 45 min at 4˚C. The
supernatant was collected and used as the total cell lysate.
Measurement of intracellular ROS
Microscopic Assessment of ROS
For ROS level measurement or oxidative stress detection NSCLC cells were cultured until
80% confluence and seeded on coverslips in 24 well plates in 500 μL of RPMI medium at a
cell density of approximately 105 cells/well. The cells were treated with emodin at different
dilutions ( 0, 1, 10, 25, 40, 50 and 75 μM ) in DMSO (vehicle < 0.5% volume) for 24, 48 and
72 h. CCCP was used as a positive control at a concentration of 50 µM, for 10 min. Post-
treatment, the media above the coverslips was aspirated and 300 µl of fresh media containing
5 μM CellROX™ Deep Red Reagent, Sub-Cellular Localization: Cytoplasm (Thermo Fisher
Scientific), (working concentration from 2.5 mM CellROX™ Deep Red Reagent stock
solution) was added to each well. The 24 well plate containing coverslips was incubated at
37°C in humidified 5% CO2 chamber for 30 min. Post incubation, the cells were washed (2
times) with PBS (pH 7.4) and fixed with 4% paraformaldehyde in PBS for 20 min. After
fixing, the cells were rinsed with PBS and stained with a 2µg/ml working Hoechst staining
solution from a Hoechst 10 mg/mL stock in PBS for 10 min at room temperature, in dark.
The cells were washed (2 times) with PBS and the coverslips were mounted on DABCO
(Sigma Aldrich) Mounting Medium (containing 1% DABCO (w/v) in 80% glycerol and 20%
PBS). The coverslips were acquired at absorption/emission maxima of 644/665 nm and
analyzed with a Nikon Real Time Laser Scanning Confocal Microscope at 60X magnification
with supporting software.
Similarly, staining was done with CellROX™ Green Reagent (Thermo Fisher Scientific), for
oxidative stress detection having a Sub-Cellular Localization: at the level of Mitochondria
and Nucleus.
Fluorometric Assessment of ROS
For ROS level measurement, NSCLC cells were cultured until 80% confluence, then seeded
in 96 well plates in 100 μL of RPMI medium at a cell density of approximately 103 cells/well.
The cells were treated with emodin at different dilutions ( 0, 1, 10, 25, 50, 75, 100 and 200
μM ) in dimethyl sulfoxide (DMSO), DMSO vehicle < 0.5% volume) for 24, 48 and 72 h.
CCCP was used as a positive control at a concentration of 50 µM, for 10 min. Post-treatment,
the media was aspirated and 100 µl of fresh serum free media containing 5 μM CellROX™
Deep Red Reagent, Sub-Cellular Localization: Cytoplasm (Thermo Fisher Scientific),
(working concentration from 2.5 mM CellROX™ Deep Red Reagent stock solution) was
added to each well. The 96 well plate was incubated at 37°C in humidified 5% CO2 chamber
for 30 min. After incubation the CellROX™ Deep Red Reagent dye fluorescence was
acquired with a 96-well ELISA plate-reader (2300 EnSpire Multimode Plate Reader,
PerkinElmer) using wavelengths of absorption/emission maxima of 644/665 nm. The staining
was performed in triplicate and each measurement contained three parallels (n=9).
Similarly, staining was done with CellROX™ Green Reagent (Thermo Fisher Scientific), for
oxidative stress detection having a Sub-Cellular Localization: at the level of Mitochondria
and Nucleus, at absorption/emission maxima of 485/520 nm.
DNA Fragmentation Analysis
NSCLC cells were seeded in 6 well plates at a cell density of approximately 105 cells/well
and treated with emodin at different dilutions (0, 25, 50 and 75 μM) in DMSO (vehicle <
0.5% volume) for 24, 48 and 72 h. Post treatment the adherent and floating cells were
collected and washed with PBS. The cell pellet was used to extract the genomic DNA using
Genomic DNA Miniprep Kit (MDI membrane technologies) as per manufacturer’s protocol.
DNA was then resuspended in Tris EDTA buffer (pH 8.0). The concentration of the genomic
DNA was determined by measuring the absorbance A260 in NanoDrop Spectrophotometer
and equal concentration of the obtained genomic DNA from each sample was loaded onto an
1.2 % agarose gel, containing ethidium bromide (0.1 mg/ml), electrophoresed at 80 V. DNA
fragmentation pattern was observed using the Syngene G:Box Gel Doc.
Indirect Immunofluorescence
For confocal microscopy, NSCLC cells were seeded on coverslips, placed in 24-well plates at
a cell density of approximately 104 cells/well and the cells were treated with emodin at 50
μM dilution in DMSO (vehicle < 0.5% volume) or vehicle alone for 24, 48, 72 h; following
which the cells were washed (2 times) with PBS (PBS) and fixed with 4% paraformaldehyde
in PBS for 20 min. These fixed cells were then washed with PBS and permeabilization was
done with 0.5% Triton-X-100 for 5 min. The PBS washed slides were then incubated for 1 h
with 5% BSA (bovine serum albumin) in PBS following which cells were stained with
various primary antibodies for 3 h. After rinsing with PBS, the coverslips were incubated
with Secondary Antibody for 1h (diluted 1:400) in 10% BSA in PBS. After PBS wash, the
DNA was counterstained with a working Hoechst staining solution at a dilution of 1:2000
(Hoechst 10 mg/mL stock in PBS) at room temperature for 10 min and the coverslips were
mounted on 1,4-diazabicyclo [2.2.2] octane (DABCO) (Sigma Aldrich) Mounting Medium
(containing 1% DABCO (w/v) in 80% glycerol and 20% PBS). The Images were acquired
and analysed with a Nikon Real Time Laser Scanning Confocal Microscope at 60X
magnification with supporting software.
Bacterial Expression and purification of MTH-1
NUDT1 was a gift from Nicola Burgess-Brown (Addgene plasmid # 74660). The plasmid
74660 for NUDT1 gene (containing N-terminal His-tagged MTH1) was obtained as a
bacterial stab of DH5α cells. The stab was revived, cells were used to prepare glycerol stocks
and the NUDT1 plasmid was extracted using pDNA Extraction kit (MDI membrane
technologies) and plasmid purity was determined by running on an agarose gel. The plasmid
was then transformed into Escherichia coli BL21-CodonPlus competent cells and plated on
LB Kanamycin agar plates to obtain single colonies. The transformed cells were grown in
100 ml cultures and Plasmid purification was done to give for sequencing and confirmation
of the NUDT1 gene. Sequence homology with NUDT1 gene was obtained. The BL21-
CodonPlus clone was grown in culture volumes of 100 ml in baffled flasks at 37 °C at 200
rpm. Once A600 nm reached 0.8, isopropyl-β-D-thiogalacto pyranoside (IPTG) (1mM) was
added and protein expression profile was studied for 1h and 3h at 37˚C and overnight at
18˚C. Since maximum protein was obtained in the overnight cultures at 18˚C, large-scale
expression and purification of MTH1 was done using the same protocol. The His-tagged
MTH1 protein was purified from the bacterial lysate using Nickel- nitrilotriacetic acid (Ni-
NTA Agarose) affinity chromatography Resin ( 30210, Qiagen, Germany ). Ni-NTA column
was washed with buffer A (10mM Tris-HCl pH 7.4, 1mM DTT, 500mM NaCl) and protein
was eluted by buffer A fortified by 150mM imidazole. Samples from each purification step
was analysed using SDS-PAGE followed by Coomassie staining. The concentration of the
purified protein was determined with a NanoDrop Spectrophotometer by absorbance A280
measurement. Pure protein was stored at −80 °C for further experiments.
Circular Dichroism Spectroscopy
The CD spectra of MTH1 was studied on a JASCO J-815 spectro-polarimeter (JASCO
Corporation, Tokyo, Japan) using a 1 mm path length cylindrical quartz cell. The MTH1
secondary structure was monitored in the region between 190 and 260 nm (far UV). An
average of three consecutive spectral scans was averaged and corresponding blank buffer was
subtracted. Data was expressed as the mean residue molar ellipticity (MRME) (deg
cm2/dmol), which is defined as the CD corrected for concentration and expressed by the
equation:
Where M is called as the molecular weight of the protein, ϴλ is the CD ellipticity (mdeg), d is
the path length (cm), C is the protein concentration (mg/mL) and r is defined as the number
of amino acid residues in the protein. The secondary structure was calculated using the K2D2
software (http://cbdm-01.zdv.uni-mainz.de/~andrade/k2d3/) for the analysis of the spectrum
obtained to quantify the content of α helix, β sheets and random coils [20].
SUPPLEMENTARY INFORMATION
Antibodies used: mouse anti - p53BP1 (pS25) (sc-135748, Santa Cruz Biotech), mouse anti -
pATM (pS1981) (sc47739, Santa Cruz Biotech), mouse anti - DNA-PKcs (sc-390698, Santa
Cruz Biotech), mouse anti - α-tubulin (sc-8035, Santa Cruz Biotech), mouse anti - β-Actin (
sc-47778, Santa Cruz Biotech), mouse anti - survivin ( sc-17779, Santa Cruz Biotech), rabbit
anti - Bax ( sc-493, Santa Cruz Biotech), mouse anti - Bcl-2 ( sc-7382, Santa Cruz Biotech),
mouse anti-Cdk2 ( sc-6248, Santa Cruz Biotech), rabbit anti - Cdk4 ( sc-601, Santa Cruz
Biotech), rabbit anti - cyclin B1 ( sc-752, Santa Cruz Biotech), rabbit anti - cyclin D1 ( sc-
753, Santa Cruz Biotech), mouse anti p - 21 ( sc-6246, Santa Cruz Biotech), mouse anti -
Integrin β1 ( sc-8978, Santa Cruz Biotech), mouse anti - vimentin ( sc-6260, Santa Cruz
Biotech), goat anti-mouse IgG-HRP ( sc-2005, Santa Cruz Biotech), goat anti-rabbit IgG-
HRP( sc-2004, Santa Cruz Biotech), rabbit anti-p53 pS15 ( 9284, Cell Signaling
Technologies), rabbit anti-Caspase-3 (full length and cleaved) ( 9662, Cell Signaling
Technologies), rabbit anti-PARP (full length and cleaved) ( 9542, Cell Signaling
Technologies), Goat anti-Mouse IgG Alexa Fluor-488 Secondary Antibody (A11001,
Thermo Fisher Scientific), Goat anti-Rabbit IgG Alexa Fluor-488 Secondary Antibody
(A11008, Thermo Fisher Scientific).
SUPPLEMENTARY FIGURES
A
B
C
Supplementary Figure 1: Measurement of cell morphology by phase-contrast microscopy. A, B and C are photographical
representation of the visual inhibition caused by increasing emodin treatment (0, 25, 50 and 75 μM) at 24, 48 and 72 h, as
measured by phase-contrast for NCI-H-460 (Large cell carcinoma), A-549 (adenocarcinoma) and NCI-H-520 (squamous cell
carcinoma) cell lines respectively.
0
5
10
15
20
25
30
35
40
45
0 25 50 75 0 25 50 75 0 25 50 75
24 H 48 H 72 H
Nu
mb
er o
f A
po
pto
tic
cells
emodin concentration (µM)
A549 NCI-H-460 NCI-H-520
A
B
Supplementary Figure 2: A: Confocal microscopic images of NSCLC cell lines stained with Hoechst 33342 dye, showing
disrupted nuclear morphology. B: Count of the number of apoptotic cells, in 50 frames for each sample as observed under
the confocal microscope.
A
Merged Nuclei Annexin PI
Control 50 µM (24hrs) 50 µM (48hrs) 50 µM (72hrs)
B
Supplementary Figure 3: A: Confocal microscopic images of NCI-H-520 cell line stained with Annexin V-FITC and PI
dyes, showing increasing green fluorescence (Annexin positivity) and red fluorescence (PI positivity). B: Graphical
representation of the mean fluorescent intensity/ nuclei indicating an increase in the fluorescence signal of apoptotic cells
with increasing emodin treatment for 24h, as observed under the confocal microscope.
A
B
Supplementary Figure 4: A: Protein level expression profiles of p-21 and p65 NFκβ respectively. B: The graphical
representation of the protein levels quantified by Image-J software. Data shown as mean ± S.E.M. of three independent
experiments.
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
0 25 50 75me
an
in
ten
sity
/nu
cle
iemodin concentration (µm)
annexin V PI
0
1
2
3
4
5
0 50µM Em 0 50µM Em 0 50µM Em
24h 48h 72 h
p21
0
0.5
1
1.5
0 50µM Em 0 50µM Em 0 50µM Em
24h 48h 72 h
p65 NFκβ
Supplementary Figure 5: Purification of MTH1 protein. A: MTH1 plasmid run on agarose gel for the determination of
plasmid purity. Lane 1: MTH1 plasmid. B: BL-21 protein expression study on SDS page gel where Lane 1: un-induced
@37°C for 1 h, Lane 2: IPTG induced @37°C for 1 h, Lane 3: un-induced @37°C for 3 h, Lane 4: IPTG induced @37°C for
3 h, Lane 5: un-induced @18°C overnight and Lane 6: IPTG induced @18°C overnight. C: the BL-21 protein purification
study on SDS page gel where Lane 1: Total Cell Lysate, Lane 2: Cell Pellet, Lane 3: Lysate supernatant, Lane 4: Lysate
Flow Through, Lane 5: Wash, Lane 6: Elution 1, Lane 7: Elution 2, Lane 8: Elution 3 and Lane 9: Elution 4. D: P-lyse
protein expression study on SDS page gel where Lane 1: un-induced @37°C for 1 h, Lane 2: IPTG induced @37°C for 1 h,
Lane 3: un-induced @37°C for 3 h, Lane 4: IPTG induced @37°C for 3 h, Lane 5: un-induced @18°C overnight and Lane 6:
IPTG induced @18°C overnight. E: Rosetta protein expression study on SDS page gel where Lane 1: Lane 1: un-induced
@37°C for 1 h, Lane 2: IPTG induced @37°C for 1 h, Lane 3: un-induced @37°C for 3 h, Lane 4: IPTG induced @37°C for
3 h, Lane 5: un-induced @18°C overnight and Lane 6: IPTG induced @18°C overnight. The data shown is representative of
three independent experiments carried out under the same conditions.
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