Suprafenacine, an Indazole-hydrazide agent, target Cancer cells through microtubule destabilization

Article abstract of DOI:10.1371/journal.pone.0110955

Microtubules are a highly validated target in cancer therapy. However, the clinical development of tubulin binding agents (TBA) has been hampered by toxicity and chemoresistance issues and has necessitated the search for new TBAs. Here, we report the identification of a novel cell permeable, tubulin-destabilizing molecule - 4,5,6,7-tetrahydro-1H-indazole-3- carboxylic acid [1p-tolyl-meth-(E)-ylidene]-hydrazide (termed as Suprafenacine, SRF). SRF, identified by in silico screening of annotated chemical libraries, was shown to bind microtubules at the colchicine-binding site and inhibit polymerization. This led to G2/M cell cycle arrest and cell death via a mitochondria-mediated apoptotic pathway. Cell death was preceded by loss of mitochondrial membrane potential, JNK - mediated phosphorylation of Bcl-2 and Bad, and activation of caspase-3.Intriguingly, SRF was found to selectively inhibit cancer cell proliferation and was effective against drug-resistant cancer cells by virtue of its ability to bypass the multidrug resistance transporter P-glycoprotein. Taken together, our resultssuggest that SRF has potential as a chemotherapeutic agent for cancer treatment and provides an alternate scaffold for the development of improved anti-cancer agents.

Full text of DOI:10.1371/journal.pone.0110955

Suprafenacine, an Indazole-Hydrazide Agent, Targets
Cancer Cells Through Microtubule Destabilization
Bo-Hwa Choi1, Souvik Chattopadhaya1, Le Nguyen Thanh2, Lin Feng1¤, Quoc Toan Nguyen1, Chuan
Bian Lim1, Amaravadhi Harikishore1, Ravi Prakash Reddy Nanga1, Nagakumar Bharatham1, Yan Zhao1,
Xuewei Liu2, Ho Sup Yoon1,3
*
1 Division of Structural Biology and Biochemistry, School of Biological Sciences, Nanyang Technological University, Singapore, Singapore, 2 Division of Chemistry and
Biological Chemistry, School of Physical & Mathematical Sciences, Nanyang Technological University, Singapore, Singapore, 3 Department of Genetic Engineering, College
of Life Sciences, Kyung Hee University, Yongin-si, Gyeonggi-do, Republic of Korea
Abstract
Microtubules are a highly validated target in cancer therapy. However, the clinical development of tubulin binding agents
(TBA) has been hampered by toxicity and chemoresistance issues and has necessitated the search for new TBAs. Here, we
report the identification of a novel cell permeable, tubulin-destabilizing molecule - 4,5,6,7-tetrahydro-1H-indazole-3-
carboxylic acid [1p-tolyl-meth-(E)-ylidene]-hydrazide (termed as Suprafenacine, SRF). SRF, identified by in silico screening of
annotated chemical libraries, was shown to bind microtubules at the colchicine-binding site and inhibit polymerization. This
led to G2/M cell cycle arrest and cell death via a mitochondria-mediated apoptotic pathway. Cell death was preceded by loss
of mitochondrial membrane potential, JNK - mediated phosphorylation of Bcl-2 and Bad, and activation of caspase-3.
Intriguingly, SRF was found to selectively inhibit cancer cell proliferation and was effective against drug-resistant cancer
cells by virtue of its ability to bypass the multidrug resistance transporter P-glycoprotein. Taken together, our results
suggest that SRF has potential as a chemotherapeutic agent for cancer treatment and provides an alternate scaffold for the
development of improved anti-cancer agents.
Citation: Choi B-H, Chattopadhaya S, Thanh LN, Feng L, Nguyen QT, et al. (2014) Suprafenacine, an Indazole-Hydrazide Agent, Targets Cancer Cells Through
Microtubule Destabilization. PLoS ONE 9(10): e110955. doi:10.1371/journal.pone.0110955
Editor: Ben C.B. Ko, The Hong Kong Polytechnic University, Hong Kong
Received October 10, 2012; Accepted September 26, 2014; Published October 29, 2014
Copyright: ß 2014 Choi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by Ministry of Health Singapore Grant NMRC1177/2008. The funders had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* Email: hsyoon@ntu.edu.sg
¤ Current address: Perinatal Research Labs, Med Surge 1, Bldg 810, University of California Irvine, Irvine, California, United States of America
Most of the microtubule-binding drugs identified so far have
been isolated from either plants or marine organisms during large-
scale screens of natural products. Microtubule-targeted anti-
Introduction
Microtubules - long, filamentous, tube shaped polymers –
mediate important roles in cellular signaling, transport of cargos,
mitotic drugs are usually classified into two groups – microtubule
establishment of cell polarity, maintenance of cell shape, cellular
migration and cell division [1,2]. Composed of a- and b-tubulin
heterodimers bound in a head-to-tail manner, microtubules are
destabilizing agents like vinca alkaloids, colchicine and microtu-
bule-stabilizing agents like paclitaxel and docetaxel. Though the
taxanes and vinca alkaloids are still administered for a wide range
not simple equilibrium polymers; instead they are highly dynamic
structures and the rapid assembly and disassembly dynamics is
crucial, in large part for their cellular functions. Not surprisingly,
microtubule polymerization is subject to tight spatial and temporal
regulation and this is achieved at several levels including (1)
transcription of different tubulin isotypes having different func-
tions; (2) by regulating a/b - tubulin ratios and heterodimer
folding (3) through various post-translational modifications of
tubulin, that in turn, alters microtubule localization and/or its
interaction with signaling pathways and (4) via interaction with
microtubule-associated proteins (MAPs) like dynein and kinesin
motor proteins, stathmin, TOG, EB1, dynactin 1, RAC1 etc [3–
5]. Paradoxically, the same dynamic nature of microtubules also
makes them exquisitely sensitive to inhibitors. By disrupting the
finely tuned behavior of microtubules, tubulin-binding drugs
interfere with the process of cell division and have proved to be
highly effective in cancer patients [6].
of cancers and are often integrated into combination chemother-
apy regimens [7,8], the current suite of tubulin-binding drugs has
several drawbacks. When compared to other anticancer drugs,
microtubule-binding drugs are structurally complex, chemically
diverse and have low solubility. Furthermore, the active drugs
occur in only minute amounts in nature and the scarcity of their
natural sources has severely hampered their clinical development.
Though this issue was addressed by the development of partial or
total synthesis methods [9] and via metabolic engineering of
pathway intermediates [10], the problem still persists where
development of new microtubule-binding compounds are con-
cerned. Another drawback is drug resistance caused by mutations
and/or expression of different tubulin isotypes like bIII-tubulin.
Drug resistance may also stem from the overexpression of drug-
efflux pumps, including the multidrug resistance transporter P-
glycoprotein (P-gp) or multidrug-resistance associated protein
(MRP) [11]. Patients being administered with microtubule-binding
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Suprafenacine as a Microtubule Destabilizing Agent
agents tend to suffer from peripheral axonal neuropathy that limits
the tolerable dose [12]. Despite these limitations, several anti-
mitotic drugs with diverse binding sites on tubulin are in various
stages of clinical development. The armamentarium of microtu-
bule-targeted agents with improved pharmacodynamic and
pharmacokinetic profiles, minimal neurological toxicity and broad
spectrum efficacy, continues to grow [13–15]. Ideally, such leads
should also be devoid of P-gp-mediated drug efflux and be
amenable to facile chemical synthesis approaches.
Cell Culture
Human cancer cell lines (HeLa, MDA-MB-231, A549, HCT15,
Jurkat, PC12 and SH-SY5Y) and human fibroblast cell lines
(CCL-116 and WI-38) were obtained from American Type
Culture Collection (ATCC, Rockville, MD, USA). SNU16,
human stomach cancer cell line, was obtained from Korean Cell
Line Bank (KCLB, Seoul, Korea). The human epithelial primary
cell line F10, and the parental cell line for the drug-resistant
mutants, KB-3-1, and multidrug-resistant line, KB-V1 were a
generous gift from Dr. Peter Dro¨ge (NTU, Singapore) and Dr. M.
Gottesman (NCI, Bethesda, MD), respectively [16]. Cell lines were
grown in either Dulbecco’s Modified Eagle Medium (DMEM) or
RPMI 1640 containing 10% fetal bovine serum, 1% penicillin/
streptomycin and maintained at 37uC in a humidified 5% CO2
chamber. For KB-3-1 and KB-1V, the media was supplemented
with an additional 1 mM sodium pyruvate and 10 mg/mL
vinblastine, respectively. All resistant lines were incubated in
drug-free media prior to cellular proliferation assays.
In this work we report a novel compound based on the indazole
scaffold, 4,5,6,7-tetrahydro-1H-indazole-3-carboxylic acid [1p-
tolyl-meth-(E)-ylidene]-hydrazide (Suprafenacine or SRF), that
shows potent anticancer properties. We discuss the identification
of SRF using in silico high-throughput screening approach, its
chemical optimization and elucidation of its microtubule binding
mode. SRF destabilizes microtubules leading to cell cycle arrest in
the G2/M phase and cell death by apoptosis. Furthermore, we
show that SRF can bypass the P-glycoprotein transporter,
specifically targets cancer cells and is effective against several
different cancer types.
Cell Cycle Analysis
Cell cycle progression was monitored using DNA flow
cytometry. Cells were seeded at a density of about 26105 cells/
well in a 24-well plate. When monolayers were 50–70% confluent,
cells were treated with drugs as indicated. After 24 h treatment,
cells were trypsinized, washed with PBS and fixed in 80% ethanol
for 1 h at 220uC. The fixed cells were resuspended in propidium
iodide (PI) staining buffer (10 mM Tris-HCl pH 8.0, 10 mM
NaCl, 50 mg/mL PI, 10 mg/L RNase A, 0.1% NP-40) for 30 min
at 4uC in the dark. The DNA content was determined on a
FACSCalibur System (BD Biosciences, San Jose, CA). For each
analysis, 10,000 cells were counted and the percentage of cells in
each phase was calculated using ModFit LT software.
Materials and Methods
Chemicals and Antibodies
Nocodazole, paclitaxel (Taxol), vinblastine, and colchicine were
purchased from Sigma-Aldrich (St. Louis, MO). SRF was
purchased from ChemDiv while SB203580, PD98059 and
SP600125 were from Calbiochem (San Diego, CA). Antibodies
were obtained from the following companies: vimentin, a- and b-
tubulin, JNK-1, Mdr-1, Bcl-2, pBcl-2 (T56), pBcl-2 (S70), pBcl-2
(S87) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA); pp38,
pERK1/2, pJNK (Cell Signaling Technology, Denver, MA);
monoclonal anti P-glycoprotein (MDR) (Sigma, USA) and
monoclonal actin antibodies were from BD Pharmingen (San
Diego, CA). [3H]vinblastine (specific activity, 11.6 Ci/mmol) and
streptavidin-coated yttrium silicate scintillation proximity assay
(SPA) beads were purchased from GE Healthcare (Buckingham-
shire, UK). [3H]colchicine (specific activity, 80.4 Ci/mmol) was
obtained from PerkinElmer (Boston, MA, USA).
Apoptosis Assays
Apoptosis was monitored using annexin V-propidium iodide
(PI) double staining method. Cells were stained using the Annexin
V-FLOUS staining kit (Roche Applied Science, Indianapolis, IN)
according to the manufacturer’s instructions. Cells were analyzed
by a BD LSR II flow cytometer (BD Biosciences, San Jose, CA)
using the 488 nm blue laser for excitation and a 505LP mirror-
530/30 BP filter and 550LP mirror-575/26 BP filter combinations
Figure 1. SRF - a novel tubulin-binding agent that depolymerizes microtubules. (A) Chemical structure of SRF. (B) Effect of SRF on
microtubule polymerization in vitro. Purified tubulin was incubated at 37uC in the absence (DMSO) or presence of drugs like taxol (3 mM), nocodazole
(10 mM), SRF and absorbance was measured at 420 nm every 1 min over a 60 min period. SRF inhibited microtubule polymerization in a
concentration-dependent manner as was indicated by a decrease in absorbance with time. (C) Confocal micrographs of HeLa cells exposed to SRF,
taxol and nocodazole. Cells were labeled with FITC-conjugated anti-tubulin antibody. SRF treatment completely destroys the intricate microtubule
network. Scale bar = 10 mM.
doi:10.1371/journal.pone.0110955.g001
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Suprafenacine as a Microtubule Destabilizing Agent
Table 1. Effect of SRF on viability of different cancer cell types.
IC50
Cell Line
Type
SRF
nM
Paclitaxel
nM
HeLa
Cervical adenocarcinoma
T-cell lymphoma
200.268.2
210.362.8
381.2628.9
252.1675.9
227.765.3
83.0617.6
237.865.5
206.763.3
6.860.2
Jurkat
5.460.1
SNU-16
MDA-MB-231
A549
Gastric carcinoma
2.960.0
Breast adenocarcinoma
Lung carcinoma
66.9639.2
15.360.9
5241.16365.6
437.2670.1
6.760.1
SH-SY5Y
HCT-15
KB-3-1
Neuroblastoma
Colorectal adenocarcinoma
Cervical adenocarcinoma
doi:10.1371/journal.pone.0110955.t001
to detect fluorescein and PI fluorescence, respectively. For each
sample, 10,000 events were collected.
SRF in the presence or absence of the JNK inhibitor, SP600125
and incubated overnight at 37uC in a 5% CO2 humidified
atmosphere. After cells were harvested, pellets were washed with
ice-cold PBS and resuspended in Cell Lysis Buffer. Freeze-thaw
method was used to lyse cells and the lysates were kept on ice for
15 min. Following clarification of the lysates by centrifugation (15,
000 g, 20 min, 4uC), the supernatant was used to determine
Caspase-3 activity. In a 96-well plate, 2 ml (10 mM stock) of Ac-
DEVD-pNA substrate was added to the reaction mixture
containing cellular lysate, DTT, Caspase Assay Buffer and
deionized water in a final volume of 100 ml. Plates were incubated
overnight at 20–22uC for 4 h and the absorbance was measured at
405 nm using a Benchmark Plus microplate reader (Bio-Rad,
Hercules). Untreated cell extracts were used as control for the
experiments.
Mitochondrial Membrane Potential Measurements
Changes in the mitochondrial membrane potential was detected
using the Mitochondrial Membrane Potential Detection Kit
(Stratagene, Cedar Creek, TX) and based on the use of the
cationic dye, 5,59,6,69-tetrachloro-1,19,3,39- tetraethylbenzimida-
zolylcarbocyanine iodide (commonly known as JC-1). JC-1 forms
fluorescent red aggregates in mitochondria of intact cells while in
apoptotic cells, collapse of the mitochondrial potential causes JC-1
to remain in the cytoplasm in its monomeric green form. Briefly,
cells were treated with SRF, in the presence or absence of JNK-
inhibitor SP600125, and incubated overnight. Cells were then
trypsinized, washed with PBS and pelleted (400 g, 5 min, RT).
Next, pellets (16106 cells/sample) were resuspended in 500 ml of 1
X JC-1 staining solution and incubated for 15 min at 37uC in a
5% CO2 humidified atmosphere. After 2X washes with assay
buffer, cells were resuspended in final volume of 500 ml of assay
buffer. Fluorescence intensities were measured on a BD FACS-
Calibur System (BD Biosciences, San Jose, CA) and the CellQuest
software was used for data analysis. Loss of mitochondrial
potential was measured as the ratio of the red-to-green fluores-
cence; compared to untreated cells, this ratio will decrease in
apoptotic cells.
Immunofluorescence Microscopy
For immunohistochemistry, cells were fixed with 3.7% PFA
followed by incubation in a blocking solution (5% BSA in PBS) for
30 min at 4uC. After 3X PBS washes, cells were permeabilized
with 0.2% Triton X-100, washed 3 X with PBS and then
incubated overnight with a- or b-tubulin antibodies at 4uC. After
washing, samples were incubated with AlexaFluor 488-conjugated
secondary antibody (Molecular Probes, Eugene, Oregon) for 1 h
at room temperature. Slides were mounted using ProLong Gold
anti-fade solution (Molecular Probes, Eugene, Oregon) containing
DAPI and visualized at 60X magnification on a Zeiss LSM
META510 confocal laser scanning microscopy. All post acquisi-
tion image processing and analysis was done using the Meta-
Morph software (Molecular Devices, LLC, Sunnyvale, CA).
Caspase Activation Assay
Caspase-3 activity was measured using the CaspACE Assay
System (Promega, Madison, WI) according to the vendor’s
protocol. Briefly, 26105 cells/well were seeded in a 6-well plate.
When monolayers were 50–60% confluent, cells were treated with
Table 2. Comparison between the effect of SRF and other microtubule binding agents on viability of drug-resistant cell types.
IC50
Cell Line
Resistant Type
SRF
nM
Paclitaxel
nM
Colchicine
nM
Vinblastine
nM
KB-3-1
KB-V1a
Parental
206.763.3
215.763.6
6.760.1
18.860.3
0.760.0
MDRq
7692.66151.4
777.1636.4
256.9642.9
aThe KB-V1 is a mutant of the parental cell line KB-3-1 and resistant to vinblastine.
doi:10.1371/journal.pone.0110955.t002
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Figure 2. SRF binds microtubules at the colchicine-binding site. (A) SRF competes with colchicine for binding to microtubules as was
determined using competition-binding scintillation proximity assay (SPA). In comparison to control (no competitor), SRF decreased colchicine
binding by 2.5-fold. Data are shown as means 6 SEM. *P,0.05, **P,0.01 versus control. (B) SRF and vinblastine have distinct binding sites on tubulin
as SRF does not compete with vinblastine. Results shown are mean (6 SD) of three independent experiments. Data are shown as means 6 SEM. *P,
0.05 versus control. (C–E) Cartoon representation of binding mode of colchicine (C) and SRF (D) to tubulin. The binding mode of drugs to tubulin was
examined by docking studies using molecular docking program GOLD (Genetic Optimization for Ligand Docking, Cambridge Crystallographic Data
Centre, UK) [Methods S1]. The compounds (colchicine or SRF) were docked into the colchicine-binding pocket of tubulin using the crystal structure of
the tubulin-colchicine: stathmin-like domain [PDB code: 1SA0]. H-bond interactions between the indazole ring of SRF and N349 and K352 of b-chain
(green) as well as the T179 and V181 of a-chain (blue) are highlighted. Panel E shows overlay of SRF and colchicine highlighting the difference in
binding patterns of these molecules.
doi:10.1371/journal.pone.0110955.g002
CO) was dissolved in G-PEM buffer (80 mM PIPES pH 6.8,
2 mM MgCl2, 0.5 mM EGTA, 1 mM GTP and 10% glycerol) to
a final concentration of 0.8–1 mg/ml. [3H]colchicine or [3H]vin-
In Vitro Tubulin Polymerization Assay
Tubulin polymerization was measured in vitro using the
Tubulin Polymerization Assay kit (Cytoskeleton, Denver, CO).
blastine (final concentration of 50 nM and 100 mM, respectively),
50 mM of SRF and 1.0 mg of biotin-labeled tubulin was mixed in a
final reaction volume of 100 ml of G-PEM buffer and incubated
for 45 min at 37uC. Streptavidin SPA beads (80 mg/well) was
added to each reaction mix and incubated for an additional
30 min at 4uC. The radioactive counts were measured using a
Wallac Microbeta scintillation counter (PerkinElmer, Waltham,
MA). For control reactions, SRF was omitted from the reaction
mix.
Tubulin was dissolved in Buffer 1 (80 mM PIPES, 2 mM MgCl2,
0.5 mM EGTA pH 6.9, 10 mM fluorescent reporter, 1 mM GTP)
to a final concentration of 10 mg/ml. To assay tubulin polymer-
ization, a mixture containing 85 ml of tubulin (final concentration
2 mg/ml), 4.4 ml GTP (stock 100 mM), 280 ml of Buffer 1 and
75 ml of Tubulin Glycerol Buffer (80 mM PIPES, 2 mM MgCl2,
0.5 mM EGTA, 60% glycerol, pH 6.9), was made and kept on ice.
Next, 5 ml of test compound, paclitaxel, nocodazole or control
buffer was aliquotted in each well of a half area 96-well, black, flat-
bottomed plate (Corning Costar). The plate was warmed for 1 min
in a 37uC pre-warmed plate reader. Polymerization was initiated
by pipetting 50 ml of reaction mix/well and monitored by
measuring the change in fluorescence (Ex = 360 nm;
Em = 420 nm). Measurement was done using SpectraFluor Plus
microplate reader (TECAN GmbH, Austria). Readings were taken
every minute for 1 h (total of 61 readings).
In Vivo Efficacy Studies
Severe combined immunodeficiency (SCID) female mice, 4–6
weeks of age, were obtained from Animal Resources Centre
(Murdoch, Australia). Mice were implanted subcutaneously in the
flank with 16107 HCT15 human colon adenocarcinoma cells.
The treatment started when tumor size attained a 100–200 mm3
or larger. Animals were treated intraperitoneally (i.p.) with 20 mg/
kg/dose SRF in final dosing formulation or a 0.9% saline vehicle.
Compound was given to tumor-bearing mice on days 1, 4, and 7
after staging, and tumor mass [(length6width2)/2] was determined
once a three days for 9 days.
Tubulin Competition-Binding Scintillation Proximity
Assay
This assay was performed for competition binding to the
colchicine and vinblastine binding sites on tubulin and carried out
in a 96-well plate. Biotin-labeled tubulin (Cytoskeleton, Denver,
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Suprafenacine as a Microtubule Destabilizing Agent
Figure 3. SRF-mediated microtubule depolymerization results in cell cycle arrest at G2/M phase. (A) Flow cytometric analysis of the DNA
content of cells treated with SRF (10 mM), nocodazole or taxol. Shown are representative one-parameter histograms of treated cells. Like taxol and
nocodazole, SRF-mediated inhibition of microtubule dynamics resulted in cell cycle arrest at the G2/M transition phase. (B) Fluorescence micrographs
of mitotic cells that had been pre-treated with the indicated compounds. Compared to vehicle treated cells, SRF and other TBAs inhibited formation
and segregation of the mitotic spindle. Microtubules (green) were stained with FITC-conjugated anti-tubulin antibody; nucleus (blue) was stained
with DAPI. Images were acquired at 60X magnification. Scale bar = 5 mM. (C) SRF-treatment induces rapid apoptosis in proliferating cells. Following a
brief 7 hr SRF exposure (10 mM), apoptotic progression was monitored by double-staining cells with Annexin V-FITC (FL-1)/PI (FL-2). During this
period, percentage of annexin V+/PI2 (indicating early apoptosis) cells increased from 2.4% (in control) to 21.7%. The kinetics of this increment is
similar to that seen with nocodazole and taxol. Both these compounds induced apoptosis in approximately 25.4% (nocodazole) and 33.2% (taxol) of
cells within this time period. The experiments were done in triplicate (n = 3) and the data value was averaged.
doi:10.1371/journal.pone.0110955.g003
Statistical analysis
Results
All experiments were independently repeated a minimum of
three times. All quantitative data are presented as means 6 SEM.
Comparisons between two groups were analyzed via student’s t
test, and values of P,0.05 were considered to be significant.
Identification of SRF as a novel tubulin-binding small
molecule
To identify novel anticancer molecules that act via binding to
tubulin, we initiated a target-based in silico screening of small
molecules using a ChemDiv library. The initial hits identified from
the virtual screen were tested for their ability to inhibit both
Figure 4. Effect of SRF on BH-3 family members, Bcl-2 and Bad. (A) Western blot analysis of HeLa and WI-38 cell extracts treated with 10 mM
SRF for 24 hr and probed with anti-Bcl-2 monoclonal antibody. A slower migrating band corresponding to phosphorylated form of Bcl-2 is present in
HeLa extracts but absent from WI-38 lysates, showing that SRF induces selective phosphorylation of Bcl-2 in cancer cells. (B) SRF mediates Bcl-2
hyperphosphorylation. Drug treated HeLa lysates were resolved on 12% SDS-PAGE and immunoblotting was done using specific phospho-antibodies
against T56, S70 and S87 residues of Bcl-2; all these amino acids lie in the flexible loop region of the protein. (C) Time course analysis of Bad
phosphorylation induced by SRF treatment. HeLa cell lysates were analyzed by immunoblotting using anti-Bad monoclonal antibody. SRF (10 mM)
treatment altered phosphorylation status of pro-apoptotic protein Bad as indicated by the appearance of a slower migrating phospho-Bad band at
24 h. The band was absent from control (DMSO treated) cells even after 24 h.
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Figure 5. SRF cytotoxicity involves JNK kinases and proceeds via caspase-3 activation and mitochondrial membrane potential loss.
(A) SRF (10 mM) activates JNK kinase but not ERK and p38. Phosphorylation status of the JNK, ERK and p38 was probed by immunoblotting using
phospho-specific antibodies against the kinases. SRF selectively induces JNK phosphorylation [Panel (i)] without altering protein levels [Panel (ii)]. (B
and C) Pre-treatment of cells with JNK-specific inhibitor prior to SRF (10 mM) exposure abrogates Bcl-2 and Bad phosphorylation. No phosphorylation
of Bcl-2 (Panel B, Lane 4) or Bad (Panel C, Lane 3) were seen when cells were pre-treated with JNK-specific inhibitor, SP600125. Similar results were not
observed with highly selective non-competitive ERK1/2 inhibitor, PD98059 (Panel B, Lane 6) or p38 inhibitor, SB203580 (Panel B, Lane 8). (D) Inhibition
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of JNK-kinase protects cells against SRF-induced toxicity. Cell viability was determined by MTT assay and reported as percentage control. SP600125
was able to retain viability in approximately 70% cells. Data are shown as means 6 SEM. **P,0.01 versus control. (E) Cells pre-treated with SP600125
were able to overcome SRF-induced G2/M phase cell cycle blockage. Percentage cells in the different stages of cell cycle were determined by flow
cytometric analysis. Data are shown as means 6 SEM. *P,0.05; **P,0.01 versus control. (F) SP600125 treated cells retain cellular microtubule
network. Fluorescence micrographs of cells treated with SRF in the presence or absence of SP600125. Microtubules (green) and nucleus (blue) were
stained with FITC-conjugated anti-tubulin antibody and DAPI, respectively. Scale bar = 10 mM. (G) SRF induces loss of mitochondrial membrane
potential as shown by flow-cytometric analysis of cells stained with JC-1. Events were counted in the green channel. SP600125 pre-treatment
prevented cells from undergoing apoptosis as percentage of cells having fluorescence in the green channel decreased from 84.2% in SRF treated cells
to 47.8% for cells that were pre-treated with SP600125 prior to SRF (10 mM) exposure. (H) Apoptotic death mediated by SRF proceeds through
caspase-3 activation. A cleaved band corresponding to activated caspase-3 is present in SRF-treated lysates but absent from SP600125 pre-treated
lysates.
doi:10.1371/journal.pone.0110955.g005
cellular proliferation and tubulin polymerization. Of all the
compounds tested, an indazole hydrazide derivative – 4,5,6,7-
Tetrahydro-1H-indazole-3-carboxylic acid [1-(3-hydroxy-4-me-
SRF inhibits cellular proliferation of various cancer cell
types
We used a colorimetric assay to evaluate the anti-proliferative
thoxy-phenyl)-meth-(E)-ylidene]-hydrazide, was found to potently
effect of SRF on cancer cells derived from a broad spectrum of
inhibit microtubule polymerization (IC50 = 0.77 mM). To further
identify analogs with improved potencies, Structure Activity
representative tumor types – cervical adenocarcinoma (HeLa, KB-
3-1, KB-V1), T-cell lymphoma (Jurkat), gastric carcinoma (SNU-
Relationship (SAR) studies were carried out [Methods S1] and a
16), breast cancer (MDA-MB-231), lung cancer (A549), colorectal
focused library of 72 analogues was synthesized and screened for
adenocarcinoma (HCT-15) and neuroblastoma (SH-SY5Y) [Ta-
their ability to inhibit microtubule polymerization. This effort
ble 1]. All cancer cell lines tested showed susceptibility to SRF
identified the analog 4,5,6,7-Tetrahydro-1H-indazole-3-carboxylic
acid [1-p-tolyl-meth-(E)-ylidene]-hydrazide (analog 4; henceforth
with IC50 values in the submicromolar range (83–381 nM).
Interestingly, when compared with taxol, SRF exhibited higher
referred to as SRF), as a potent molecule (IC50 = 0.38 mM)
[Figure 1A].
potency towards the neuroblastoma SH-SY5Y and colorectal
adenocarcinoma HCT-15 cell lines. Furthermore, we also tested
the effect of SRF on normal cells like skin fibroblast cell line CCL-
116 and human epithelial primary cell line F10. Both CCL-116
and F10 showed greater than 80% survival following a 24 h
exposure to SRF while only 60% survival was seen when the same
Figure 6. The antitumoral effect of SRF on human colon adenocarcinoma HCT15 xenografts. SCID female mice were implanted with
16107 HCT15 human colon adenocarcinoma cells. The treatment started when tumor size attained a 100–200 mm3 or larger. Animals were treated
intraperitoneally (i.p.) with 20 mg/kg/dose SRF in final dosing formulation or a 0.9% saline vehicle. Compound was given to tumor-bearing mice on
days 1, 4, and 7 after staging, and tumor mass [(length6width2)/2] was determined once a three days for 9 days. Three tumors were observed at
different time points. Error bars represent standard error of the mean (n = 3). The data were analyzed by a one-sided Student’s t test, and values of P,
0.05 were considered to be significant. *P,0.05 versus vehicle control.
doi:10.1371/journal.pone.0110955.g006
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Suprafenacine as a Microtubule Destabilizing Agent
Figure 7. Cartoon representing plausible mechanism of SRF mediated toxicity in cancer cells. SRF can bypass P-gp mediated drug efflux
via mechanism(s) that are currently unknown. Inside the cell, SRF binds and inhibits microtubule polymerization resulting in cell cycle arrest at the G2/
M phase. These events, in turn, activate JNK-mediated stress-response signaling cascade leading to the phosphorylation and inactivation of anti-
apoptotic proteins like Bcl-2 and Bad. Consequently, there is loss of mitochondrial membrane potential and integrity, release of cytochrome-c,
activation of caspase-3 and eventual cell death by apoptosis. Thus, SRF-mediated cell death proceeds via the intrinsic/mitochondrial apoptotic
pathway.
doi:10.1371/journal.pone.0110955.g007
cells were treated with taxol under identical conditions [Figure
S1]. Taken together, our data confirm that SRF selectively targets
cancer cells and is efficacious against several different cancer types.
increase in expression of mdr1 gene, which encodes P-glycoprotein
(P-gp), an energy-dependent efflux pump. To be clinically
relevant, SRF must be able to bypass the P-gp-mediated efflux.
We therefore determined effect of SRF on proliferation of
epidermal carcinoma cell line KB-V1, a vinblastine resistant
subline derived from the parental KB-3-1 cells [16] [Table 2].
KB-V1 cells are highly enriched in P-gp and they show cross-
resistance to colchicine and adriamycin [Figure S2]. Comparison
SRF can bypass the p-glycoprotein drug efflux pump
The clinical success of the drugs used in cancer treatment has
been hampered by the development of drug-resistance. The
phenomenon of multidrug resistance is often associated with an
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Suprafenacine as a Microtubule Destabilizing Agent
of the anti-proliferative effect mediated by SRF and taxol revealed
that SRF is 35-fold more potent than taxol against KB-V1 cell line
while both compounds showed similar efficacy against KB-3-1
cells, as these cells are not known to overexpress P-gp [17]. The
ability of SRF to bypass the P-gp probably explains why it is more
potent than taxol in the SH-SY5Y neuroblastoma and HCT-15
colorectal adenocarcinoma cells as both these cancer cell types
express elevated levels of P-gp protein [17–19].
phase in a dose-dependent manner and dramatic increase in the
G2/M population of the cells treated with 10 mM of SRF
[Figure 3A and Figure S3]. Similar effects on microtubules were
observed with nocodazole and taxol. Cell cycle arrest was
accompanied by the failure of the mitotic spindle to segregate in
rapidly dividing cells [Figure 3B]. However, no significant increase
in the G2/M phase was observed in normal cell lines (CCL-116,
WI-38 and F10) upon SRF treatment [Figure S4]. Since cell cycle
arrest invariably triggers apoptosis, we performed annexin V-PI
double staining on SRF-treated cells so as to monitor phospha-
tidylserine exposure, a signal for early apoptosis. Following a brief
7 h exposure, the population of early apoptotic cells (annexin V+/
PI2) dramatically increased from 2.4% to 21.7% [Figure 3C],
thereby indicating that SRF induces rapid apoptosis in prolifer-
ating cells.
SRF destabilizes microtubule assembly by binding to the
colchicine-binding site
To evaluate the principal mode of action of SRF, we profiled its
effect on tubulin polymerization in vitro [Figure 1B]. The results
show that SRF inhibits tubulin polymerization in a concentration-
dependent manner with an IC50 of 0.38 mM. In addition, the
microtubule-disrupting effect of SRF was compared with two
different microtubule targeted drugs, taxol, Nocodazole, known as
anti-neoplastic agents by interfering the polymerization of tubulin.
Notably, SRF affected the onset of the polymerization (nucleation
phase) and its rate (elongation phase) resulting in reduced tubulin
polymer mass. We also studied the effect of SRF on the
microtubule assembly at the cellular level using immunohisto-
chemistry. HeLa cells exposed to SRF for 24 h were fixed and
microtubules stained with a- or b-tubulin antibodies. Compared
with untreated control cells, which maintain an intricate and
organized microtubule network, SRF treatment caused a complete
destruction of this cytoskeletal network and appearance of
abundant, characteristic short bundles that were distributed all
over the cytoplasm in these cells [Figure 1C]. Similar disruption of
the microtubules was observed with nocodazole exposure whilst
taxol treatment produced shorter but denser microtubules
SRF induces apoptosis via JNK-mediated
phosphorylation of Bcl-2 and Bad
The Bcl-2 family of proto-oncogenes is master regulators of
apoptosis and function as molecular rheostats to control cellular
survival [21]. Since anti-mitotic drugs like paclitaxel, vincristine,
vinblastine, induce Bcl-2 hyperphosphorylation in the loop region
[22–26], we investigated the effect of SRF on Bcl-2. Extracts from
HeLa cells treated with 10 mM SRF for 24 h were analyzed by
immunoblotting with monoclonal anti-Bcl-2 antibody. The results
show that together with a Bcl-2, SRF-treated cells have additional
band(s) that has slower migration rate compared to Bcl-2
[Figure 4A, Upper Panel]. In contrast to HeLa cells, vehicle-
treated cells completely lacked the extra band. Likewise, the
phosphorylation state of Bcl-2 in the normal diploid lung fibroblast
cell line WI-38 remained unchanged even after 24 h SRF
treatment [Figure 4A, Middle Panel]. To confirm that the band(s)
are indeed phosphorylated forms of Bcl-2, blots were then probed
with phospho-Bcl-2 specific antibodies. SRF induced the phos-
phorylation of Bcl-2 at multiple residues (T56, S70 and S87), all of
which lie within a flexible loop region [Figure 4B]. Taken
together, these observations further reiterate the fact that SRF
selective targets cancer cells. In addition to Bcl-2, a 24 h exposure
to SRF treatment also induced the phosphorylation of the pro-
apoptotic Bcl-2 family member – Bad [Figure 4C] while Bcl-xL,
Bak and Bax remained unaffected [Figure S5].
consistent with its role as
[Figure 1C].
a microtubule-stabilizing agent
Compounds that inhibit tubulin polymerization mostly bind to
the known distinct sites, namely taxol, vinblastine, orcolchicine
sites of tubulin. A competitive-binding scintillation proximity assay
(SPA) was used to probeSRF binding site on tubulin. SRF did not
reduce specific SPA counts stimulated by conjugating the biotin-
labeled tubulin with [3H] taxol or with [3H] vinblastine, but
strongly competed for binding of [3H] colchicine to tubulin
[Figure 2A and 2B]. Next, molecular modeling and docking
studies were done to further ascertain the binding mode of SRF.
Using the crystal structure of the tubulin-colchicine: stathmin-like
domain (PDB 1SA0), both colchicine [Figure 2C] and SRF
[Figure 2D] were docked into the colchicine-binding pocket of
tubulin. Despite the structural dissimilarity, SRF shares the same
cartesian space as colchicine. Unlike colchicine, SRF lacks
hydrogen bond interactions with Cys241 of b-chain. Instead, the
indazole ring of SRF forms hydrogen-bond interaction with
Asn349, Lys352 of the b-chain as well as Val181 and Thr179 of a-
chain while the 4-methyl group of the distal phenyl ring forms
hydrophobic interactions with b-chain Lys 254. Taken together,
the docking studies reveal that though SRF and colchicine bind to
identical site on tubulin, SRF has a binding mode slightly different
from that of colchicine [Figure 2E].
The mitogen-activated protein kinases (MAPK), expressed in all
cell types, transduce signals from the cell membrane to the nucleus
in response to a variety of stimuli and also regulate a wide
spectrum of biological processes critical for cellular homeostasis
[27]. Among the different pathways mediated by MAPK family
members, the extracellular signal-regulated kinase 1 and 2
(ERK1/2), c-Jun N-terminal kinase/stress-activated protein kinase
(JNK/SAPK) and p38 MAPK, are known to be activated by
cellular stresses such as heat, osmotic shock, UV and c irradiation,
metabolic poisons, pro-inflammatory cytokines and in some
instances, by growth factors and GPCR agonists [28–31]. In fact,
it has been shown that vinblastine induces apoptosis via JNK-
mediated phosphorylation of Bcl-2 while all three MAPK
pathways (JNK, ERK and p38 kinase) are activated upon taxol
exposure [32,33]. We therefore wanted to monitor the activation
of MAPK pathway/s following treatment of HeLa cells with SRF
for 24 h. Phospho-specific antibodies against JNK, ERK1/2 and
p38 were used to selectively stain for activated form of these
kinases. While SRF treatment had no effect on the expression
levels of the kinases (data not shown), SRF induced an increase in
JNK activity as evident by the appearance of a band correspond-
ing to phosphorylated form of JNK [Figure 5A, Panel (i)]. No
change in specific activity of ERK1/2 and p38 were detected
SRF arrests cell cycle in G2/M-phase
Since microtubule dynamics has important roles in cell cycle
progression, mitotic stall may lead to various chemotherapeutic
outcomes including mitotic death, mitotic exit, apoptosis or
aneuploidy [11,20]. To study effect of SRF on cell cycle
progression, HeLa cells were treated with different concentration
of SRF for 24 h and cell cycle was monitored by flow cytometry.
SRF treatment caused a complete collapse of all the cells at the G1
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Suprafenacine as a Microtubule Destabilizing Agent
[Figure 5A, Panel (iii) and (iv)]. To further verify that activation of
JNK cascade is indeed casual to phosphorylation of Bcl-2 and Bad,
we employed specific inhibitors - SP600125, PD98059 and
SB203580 that target JNK, ERK1/2 and p38 kinase, respectively.
Prior to SRF treatment, cells were incubated with the MAPK
inhibitors at 10 mM concentrations for 1 h. While PD98035 and
SB203580 did not alter Bcl-2 phosphorylation, pre-treatment with
SP600125 completely abolished the presence of the phosphory-
lated forms of both Bcl-2 and Bad [Figure 5B and 5C]. To further
confirm the association of the activated JNK to the hyperpho-
sphorylation of Bcl-2 and Bad, the behavior of phosphorylated
form of JNK was examined prior to 24 h post-treatment, while in
parallel, the phospho-Bcl-2 and phospho-Bad were also deter-
mined after JNK inhibitor SP600125 treatment. Apparently, in
the absence of SRF, none of activated form of JNK was observed
[Figure S6A]. The result also confirmed the gradually decrease of
phosphorylated form of JNK in a time-dependent manner (8, 12,
24 hours after SRF and SP600125 treatment) [Figure S6B].
Moreover, no phosphorylated form of Bcl-2 (no shifted band) was
detected whereby about 50% decreased level of phosphorylated
form of JNK was observed within 8 hours after SRF and
SP600125 treatment. Taken together, our data suggest that the
SRF-induced apoptosis could be mediated primarily through
JNK-mediated phosphorylation of Bcl-2.
caspase-3 activity over control cells when enzymatic activity was
assessed using
a caspase-3 specific fluorogenic tetrapeptide
substrate, Ac-DEVD-pNA [Figure S6D]. In all cases, pre-
treatment of cells with the JNK inhibitor SP600125 abrogated
the caspase-3 activation thereby further underscoring the impor-
tance of JNK in SRF-mediated cell death pathway.
In vivo efficacy of SRF against colon adenocarcinoma
HCT15 xenograft
In addition to the direct cytotoxicity of SRF to a variety of
human cancer cell lines, this antitumoral effect was also examined
in animal model using human tumor xenografts in mice. SRF
activity retained substantially against HCT15 cells that expressed
high levels of P-gp, and that were relatively resistant to taxol
[Figure S2]. Thus, an anti-tumor efficacy of SRF was performed in
SCID mice bearing tumors derived from HCT15 colon adeno-
carcinoma. 16107 HCT15 cells were implanted as s.c. at one flank
per mouse [Figure 6]. Nine days after implantation, when the
well-established HCT15 xenografts were obtained with tumor size
about 100 mm3 or larger, mice were treated intraperitoneally (i.p.)
with vehicle control or with 20 mg/kg/dose SRF in final dosing
formulation. Compared with tumor growth in control mice, we
found that SRF induced a significant reduction of tumor mass by
40% on day 9 in a time-dependent manner, suggesting that SRF
potentially suppresses tumor growth in vivo.
Further evidence that the JNK signaling pathway is central to
SRF-mediated toxicity came from cell viability assays, immuno-
histochemistry and flow cytometric analysis. Pre-treatment of
HeLa cells with SP600125 resulted in a 70% increase in cell
viability when compared to cells exposed to only SRF while
PD98059 and SB203580 treatment did not show any significant
reduction in cellular toxicity [Figure 5D]. Cell cycle analysis by
flow cytometry showed that 60% of SP600125 pre-treated cells
can overcome the SRF-mediated G2/M arrest, whereas cells
treated with PD98059 and SB203580 treatment continue to
remain arrested in the G2/M phase and eventually undergoes
apoptotic death [Figure 5E and Figure S6]. Furthermore,
immunohistochemical analysis revealed that cells treated with
SP600125 were able to maintain the intricate microtubule network
that is normally destroyed upon SRF treatment [Figure 5F].
Discussion
The concept of using anti-tubulin compounds in anticancer
treatment is not new. Besides inhibiting tumor cell proliferation,
several of these tubulin-binding agents (TBA) can rapidly disrupt
the tumor vasculature [37] and have been the mainstay of
chemotherapeutic agents. However, emergence of chemoresis-
tance and their toxic neurological side effects have hampered the
clinical development of these molecules. Owing to the importance
of microtubules in cell cycle progression, microtubules continue to
remain as attractive target for anticancer drugs. In our effort to
identify new TBAs, we performed an in silico virtual screening of
chemical library against tubulin to identify an indazole hydrazide
derivative as a novel TBA. SAR analysis and lead optimization
gave us the final compound – SRF that inhibits tubulin
polymerization with submicromolar potencies. Our scintillation
proximity assay results show that SRF binds to the colchicine-
binding site on tubulin but has a binding mode distinct from that
of colchicine. Owing to its extreme toxicity, colchicine itself is not
an anticancer agent but other colchicine-domain binding drugs,
such as combretastatins, 2-metoxyestradiol (2-ME) and chalcones,
are now being actively investigated for their anticancer activities
[38]. SRF shows broad-spectrum activity as it effectively inhibits
proliferation of several different cancer cell types derived from
solid tumors and hematological malignancies, as well as neuro-
blastomas and drug-resistant [Table 1 and Table 2]. Notably,
SRF is selective against cancer cells, as it had little/no effect on
normal diploid cells like CCL-116, F10 and WI-38 [Figure S1 and
Figure S4]. The basis of this selectivity is presently not known.
SRF is not a substrate for the efflux pumps as it exhibits a similar
potency irrespective of the cell’s MDR status. SRF not only
exhibited its direct anti-proliferative activity in cell based assays
(KB-3-1 and KB-V1), but also demonstrated an anti-tumor
activity in vivo in human HCT15-implanted xenografts with an
effective dose of SRF (20 mg/kg/day in mice) [Figure 6]. This
anitumoral efficacy of SRF in animal model can be compared with
other recent tubulin polymerization inhibitors, BPR0L075 and
EM015, which were reported with effective doses of 50 mg/kg/
SRF-mediated apoptosis involves via loss of
mitochondrial membrane potential and activation of
Caspase-3
Various extracellular and intracellular stresses the trigger the
intrinsic apoptotic pathway and these signals converge mainly on
the mitochondria resulting in opening of the mitochondrial
transition pore (MTP), loss of mitochondrial transmembrane
potential and release of cytochrome c together with other pro-
apoptotic molecules [34,35]. Bcl-2 family members govern
mitochondrial integrity and therefore regulate these apoptotic
mitochondrial events [36]. Since phosphorylation of Bcl-2 and
Bad results in loss of their cellular activity, we assessed the effect of
SRF on integrity of the mitochondrial membrane. Using the JC-1
reagent in flow cytometric analysis, we found that SRF treatment
results in a time-dependent increase (from 4.8% to 84.2%) of the
JC-1 monomeric form that eventually peaks at 24 h while pre-
treatment with the SP600125 inhibitor resulted in a 10-fold
increase of green fluorescence from 4.8% to 47.8% [Figure 5G].
Furthermore, loss of mitochondrial integrity was accompanied by
caspase-3 activation as was evident from immunoblots with anti-
caspase-3 antibody. A cleaved band corresponding to activated
caspase-3 was present in extracts of cells treated with SRF while
control cells completely lacked this form [Figure 5H]. Likewise,
lysates from cells treated with SRF showed a 3-fold increase in
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Suprafenacine as a Microtubule Destabilizing Agent
day and 300 mg/kg/day in mice, respectively [13,15]. Further
studies of SRF in a variety of xenograft tumor models (for example
in KB-3-1- and KB-V1-xenografted mice) might provide insightful
information into chemotherapeutic potential of SRF as a novel
microtubule-targeting agent. Whether SRF can overcome resis-
tance mediated by tubulin mutations and/or expression of
different tubulin isotypes also needs further investigation.
results warrant further investigation of the pharmacokinetic
properties of SRF and determination of its in vivo efficacy in
animal models as the SRF scaffold holds great promise as a
prototype for the development of a new class of anti-tubulin
agents.
Supporting Information
To further evaluate that microtubules are bona fide targets of
SRF, the effect of compound at the cellular level was analyzed by
immunofluorescence microscopy. Microtubule binding by SRF
completely destroyed the cellular microtubule network causing the
microtubules to bundle indicating a decrease in the polymer mass.
Since attenuation of microtubule dynamics engages cell cycle
surveillance mechanisms to arrest cell division in mitosis, SRF-
mediated inhibition of microtubule polymerization lead to a
complete blockade of the cell cycle at the G2/M phase and
eventual cell death by apoptosis. The anti-tumor effects of SRF
were shown to be associated with the microtubule depolymeriza-
tion and apoptosis induction [Figure S7]. In addition, SRF-
mediated cell death had all the canonical hallmarks of apoptosis as
was evident from loss of mitochondrial membrane potential and
concomitant activation of caspase-3. We, therefore hypothesize
that SRF triggers apoptosis via the intrinsic mitochondrial
pathway (Figure 7).
Figure S1 SRF selectively inhibits proliferation of
cancer cell types. Normal cells like skin fibroblasts CCL-116
and human primary cells F10 were treated with 10 mM of SRF for
24 hr and cell viability was measured by MTT assay. Compared
to control (untreated) cells, both CCL-116 and F10 cells showed .
80% survival when exposed to SRF while only 60% cells survived
with taxol under identical conditions. Results shown are mean 6
SEM of three independent experiments.
(PDF)
Figure S2 Expression of P-glycoprotein on different
cancer cell lines. The total cell lysates of 11 cancer cell lines
(HeLa, HCT15, MDA-MB-231, Jurkat, SNU16, SH-SY5Y,
PC12, MCF-7, A549, KB-3-1 and KB-V1) were resolved by
12% SDS-PAGE and transferred into the nitrocellulose mem-
brane for Western blot analysis. The blot was probed with
monoclonal anti-P-glycoprotein antibody. GAPDH was used as a
loading control.
The Bcl-2 protein family has pivotal roles in regulating cell
survival in part by affecting the mitochondrial compartmentaliza-
tion of cytochrome c [39]. Since SRF-treatment causes loss of
mitochondrial membrane integrity, we examined its effect on the
Bcl-2 proteins. Bak, Bax and Bcl-xL remained unaltered while Bcl-
2 was hyperphosphorylated at multiple serine and threonine
residues (T56, S70 and S87) that lie within the unstructured
flexible-loop region of the protein. Bcl-2 phosphorylation interferes
with its anti-apoptotic role as it loses its ability to heterodimerize
with Bax [40,41]. Consequently, cells arrested in the G2/M phase
become increasingly sensitized to stress signals and undergo rapid
apoptosis [22]. In addition to phosphor-Bcl-2, we also observed
the presence of the phosphorylated form of the BH3-only protein
Bad, in HeLa cell extracts treated with SRF. Bad is unique as it
serves to integrate both pro-survival and proapoptotic signals with
the net effect contributing to either cell survival or apoptosis.
Survival-promoting kinases including Akt, Rsk, p21-activated
kinase, p70S6 kinase, suppress Bad-mediated apoptosis by
inducing Bad phosphorylation at S112 and/or S136 [42–45]
and this, in turn, leads to Bad sequestration by 14-3-3 proteins
[46]. On the other hand, apoptosis-inducing kinases like Cdc2 and
JNK mediate Bad phosphorylation at S128 [47,48]. Whether SRF
induces Bad phosphorylation at S128 remains to be determined.
Furthermore, we also delineated the signaling cascade responsible
for Bcl-2 and Bad phosphorylation. Consistent with literature
reports [22,47,49,50] JNK was found to be involved in both cases
of phosphorylation. These events were abrogated when cells were
pre-treated with the JNK inhibitor SP600125 but not with
PD98058 or SB203580, which inhibit ERK1/2 and p38 kinase,
respectively. Cells pre-treated with SP600125 were also able to
overcome G2/M cell cycle blockade following SRF exposure,
retain mitochondrial membrane integrity and prevent caspase-3
activation thereby underscoring the importance of JNK signaling
in SRF-mediated cellular toxicity.
(PDF)
Figure S3 Concentration-dependent effects of SRF on
cell cycle of HeLa cells were analyzed by flow cytometer.
Cells were treated with indicated dose-dependent concentration of
SRF for 24 hours. Untreated cells were performed as negative
control. Taxol-treated cells were used as a positive control. After
drug treatments, cells were fixed with 80% ethanol and stained
with 50 mg/mL propidium iodide for 30 min at 4uC in the dark.
The DNA contents of cells were examined by flow cytometry.
Results shown are from one experiment performed in triplicate.
(PDF)
Figure S4 Effects of SRF on cell cycle progression in
different human cell lines. The percentage of the DNA
content of cells treated with SRF (10 mM) was determined by flow
cytometer. Shown are representative one-parameter histograms of
treated cells. Unlike the cancer cell lines (MDA-MB-231, KB-V1
and A549), SRF-treated normal cell lines (CCL-116, WI-38 and
F10) did not show a significant increase of cell cycle in G2/M
transition phase.
(PDF)
Figure S5 SRF does not phosphorylate Bcl-2 family
members other than Bcl-2 and Bad. Extracts of HeLa cells
treated with 10 mM of SRF for the indicated times were probed
with antibodies against Bcl-XL, Bak and Bax. Only a single band
corresponding to the full-length protein was visible in all the blots.
(PDF)
Figure S6 The effect of inhibition of JNK kinase on the
phosphorylation of Bcl-2. (A) SP600125 pre-treatment can
prevent SRF-induced JNK phosphorylation and activation
without altering protein levels. Blots were probed with phospho-
JNK and JNK specific antibodies. (B) Gradual decrease in the
phosphorylated form of JNK was determined in total HeLa cell
lysate treated with SRF and SP600125. In correlation with
changes of activated form of JNK, the phosphorylation form of
Bcl-2 was detected according to the time-scale (8, 12, 24 hours
post-treatment). Blots were probed with anti-phopho-JNK and
anti-Bcl-2 antibodies. (C) Cells treated with p38 (SB203580) and
In conclusion, the data presented herein provides compelling
evidence that the novel indazole hydrazide-based compound –
SRF, by virtue of (1) its has broad-spectrum efficacy against several
different tumor types, (2) selectivity for cancer cell types and (3)
ability to bypass multidrug resistance, has potential as an
antineoplastic drug for treatment of various types of cancer. Our
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Suprafenacine as a Microtubule Destabilizing Agent
ERK1/2 (PD98059) inhibitors are not able to overcome SRF-
induced cell cycle blockade at the G2/M phase. Cells were stained
with PI and the DNA content was analyzed by flow cytometry. (D)
Caspase-3 activity in SRF-treated cell lysate was determined using
the fluorogenic substrate Ac-DEVD-pNA. SRF induces a 3-fold
increase in enzymatic activity that is decreased in presence of
specific JNK inhibitor, SP600125. The internal protein levels were
detected by using anti-histone H3 antibody.
membranes were probed with anti-b-tubulin, anti-cleave PARP
and anti-phosphorylated Bcl-2 antibodies. The protein expression
levels of vimentin in the cell lysates was detected by anti-vimentin
antibody, which is an internal loading control.
(PDF)
Methods S1
(PDF)
(PDF)
Author Contributions
Figure S7 Molecular characterization of the anticancer
effects of SRF in HeLa cells. The cancer cells were treated
with different concentrations of SRF (0, 10, 25, 50 mM) for 24
hours. Proteins (50 mg/lane) in the cell lysates were separated by
SDS-PAGE and transferred to nitrocellulose membranes. The
Conceived and designed the experiments: BHC XL HSY. Performed the
experiments: BHC SC LNT LF AH RPRN NB CBL YZ QTN. Analyzed
the data: BHC SC LNT LF AH CBL QTN XL HSY. Contributed
reagents/materials/analysis tools: XL HSY. Wrote the paper: BHC SC
AH XL HSY.
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