Synthesis and biological evaluation of tetrahydropyridinepyrazoles ('PFPs') as inhibitors of STAT3 phosphorylation

Article abstract of DOI:10.1039/c3md00119a

The transcription factor STAT3 is constitutively overexpressed in many human tumors and hence represents a putative target for anticancer drug design. In this work, we describe the synthesis and biological evaluation of a novel chemotype, pyridine-fused p

Full text of DOI:10.1039/c3md00119a

Volume 5 Number 1 January 2014 Pages 1–108
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CONCISE ARTICLE
Mantelingu K, Andreas Bender, Rangappa KS et al.
Synthesis and biological evaluation of tetrahydropyridinepyrazoles (‘PFPs’)
as inhibitors of STAT3 phosphorylation

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CONCISE ARTICLE
Synthesis and biological evaluation of
tetrahydropyridinepyrazoles (‘PFPs’) as inhibitors of
STAT3 phosphorylation†
Cite this: Med. Chem. Commun., 2014,
5, 32
Revanna C. N,‡ab Basappa,‡cd Srinivasa V,c Feng Li,e Kodappully Sivaraman Siveen,e
Xiaoyun Dai,e Shivananju Nanjunda Swamy,f Bhadregowda D. G,a Gautam Sethi,eg
h
d
h
*
*
*
Mantelingu K, Andreas Bender and Rangappa KS
The transcription factor STAT3 is constitutively overexpressed in many human tumors and hence represents
a putative target for anticancer drug design. In this work, we describe the synthesis and biological evaluation
of a novel chemotype, pyridine-fused pyrazoles (‘PFPs’) as inhibitors of STAT3 phosphorylation. The effect
of the compounds synthesized was evaluated in cell proliferation assays of MCF-7 and HepG2 cancer cell
lines and two of the compounds tested (12g and 12k) were found to show significant activity. Both
compounds were also found to inhibit the proliferation of Hep3B, HUH-7 and PLC/PRF5 HCC cells in a
dose- and time-dependent manner. Furthermore, we established in a DNA binding assay that one of the
compounds (12g) was able to significantly inhibit the DNA binding ability of STAT3. Cytotoxicity of 12g
against PC3 cells, which do not constitutively phosphorylate STAT3, was found to be minimal, hence
lending further support for our mode-of-action hypothesis of this compound. We established for this
structure a complete inhibition of CXCL12-induced cell invasion and associated wound healing in
HCCLM3 cells, corroborating the proposed modulation of the STAT3 axis by 12g. Finally, molecular
modeling was employed to evaluate the hypothesis of PFPs to bind to the SH2 domain of STAT3. Given
the efficacy of PFPs in the biological systems studied here we propose their further evaluation in the
context of STAT3-mediated cancer therapy.
Received 19th April 2013
Accepted 26th October 2013
DOI: 10.1039/c3md00119a
www.rsc.org/medchemcomm
Signal Transducer and Activator of Transcription proteins and breast, as well as myeloma and leukemia.1,2 It is involved in
(STATs) are involved in both developmental and oncogenic the expression of a variety of genes in response to cell stimuli,
signaling pathways and comprise seven members, namely and thus plays a key role in many cellular parameters like cell
STAT1 to STAT4 and STAT6, as well as STAT5a and STAT5b growth and apoptosis, as well as multiple steps of metastasis
which are closely related. STAT3, which belongs to a mamma- including cell survival, invasion, angiogenesis, and the evasion
lian STAT family, is frequently activated in many human of tumor-cells to the endothelium.3,4 Functionally STAT3 can be
cancers, including those of liver, lung, head and neck, prostate activated by certain growth factors, cytokines and hormones
and this activation leads to intracellular phosphorylation fol-
aDepartment of Chemistry, Yuvarajas College, University of Mysore, Mysore-560005,
lowed by dimerisation. The STAT3 dimer translocates into the
nucleus, binds to DNA and promotes gene transcription,
leading in turn (when functioning as an oncogene) to cell
growth, cell survival, angiogenesis and resistance to
apoptosis.5–7 STAT3 expression is predominant in hepatocel-
lular carcinoma (HCC), a fatal liver cancer affecting 600 000
people annually and thus one of the leading causes of cancer-
related lethality.8,9
Given its functions outlined above, inhibiting the phos-
phorylation of STAT3 was proposed as a target for cancer
therapy, either directly or indirectly, and this concept was
supported by work involving viral vectors which signicantly
inhibited STAT3 signaling.10 Also, given the increasing interest
in the area, several small-molecule inhibitors of this protein
have recently been published.11–13 In order to block STAT3
indirectly the inhibition of the STAT signaling pathway by
India
bSyngene International Limited, Jigini Link Road, Bangalore-560009, India
cLaboratory of Chemical Biology, Department of Chemistry, Bangalore University,
Palace Road, Bangalore-560001, India
dUnilever Centre, Department of Chemistry, Cambridge, CB2 1EW, UK. E-mail:
ab454@cam.ac.uk
eDepartment of Pharmacology, Yong Loo Lin School of Medicine, National University of
Singapore, Singapore 117597
fDepartment of Biotechnology, Sri Jayachamarajendra College of Engineering, JSS
Technical Institutions Campus, Mysore 570006, Karnataka, India
gCancer Science Institute of Singapore, National University of Singapore, Centre for
Translational Medicine, 14 Medical Drive, Singapore 117599
hDepartment of Chemistry, University of Mysore, Manasgangotri, Mysore-560006,
India
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c3md00119a
‡ These authors contributed equally to this work.
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blocking upstream tyrosine kinases has been proposed. For and migration of cancer cells (MCF-7, HepG2, HUH-7, PLC/PRF/
example, small-molecule inhibitors of Proto-Oncogene Tyro- 5, and Hep3B cell lines) as well as inhibiting the DNA-binding
sine-Protein Kinase (SRC), Janus Kinase (JAK), Breakpoint ability of STAT3 in HCC cells.
Cluster Region Protein-V-Abelson Murine Leukemia Viral
Oncogene Homolog 1 (BCR-ABL), Fms-like tyrosine kinase 3
(FLT3), and Epidermal growth factor receptor (EGFR), have all
Results and discussion
been shown to block STAT3 or STAT5 signaling and induce
Effect of PFPs on the proliferation of cancer cells
tumor-cell apoptosis.14–16 Also some traditional medicines (such
Initially, we investigated the anti-proliferative effect of PFPs on
MCF-7 cancer cells and it was found that 12d, 12e, 12g, and 12k
were able to inhibit the proliferation of MCF-7 cells by 85.3%,
78.8%, 77.4%, and 84.1%, at 100 mM, respectively (Fig. 2).
Furthermore, 12g and 12k also inhibited the proliferation of
HCC cancer cells (HepG2) by 80% and 85.1%, respectively
(Fig. 3). These data show that 12g and 12k, where R ¼ arylated
urea and thiourea, are most potent among all PFPs tested. The
sulphonamide substituted PFPs 12d and 12e could in principle
work as prodrugs/bioprecursors, however they failed to inhibit
the proliferation of HepG2 cells in our study.
as cucurbitacin I) are known to be selective inhibitors of JAK
and hence to modulate STAT3 signaling.17
The main drawback of inhibitors of upstream tyrosine
kinases is that they have many targets (both due to kinase
inhibitor promiscuity as well as cross-talk in signaling path-
ways), which is different with compounds that target STAT3
directly. Although the selective direct inhibition by means of
small molecules is certainly not trivial some progress has
already been made in this direction. Most of those involve tar-
geting the Src Homology2 (SH2) domain of STAT3 which is
essential for both tyrosine-phosphorylation and dimerisation,
with the hope that inhibition of dimerisation also translates to
efficacy in the clinic.18 Several dimerisation disruptors of STAT3
have been reported and are in various phases of clinical devel-
MTT dye uptake assay of HCC cells
opment; still the area is a eld of active research and no We next investigated whether the most potent compounds
compounds have been approved as of yet.
against MCF-7 and HepG2 cells can also inhibit the prolifera-
Given the biological relevance of targeting STAT3, we in this tion of other HCC cells via MTT dye uptake assay as described in
work report a new class of inhibitors for the phosphorylation of detail in the Material and Methods section. Here it was found
STAT3 by pyridine-fused pyrazoles (‘PFPs’, see Fig. 1 and Table 1 that both 12g and 12k were able to inhibit the proliferation of
for structural and synthetic details) and we show that the most HepG2, HUH-7, PLC/PRF/5, and Hep3B cells in a dose- and
active compounds are able to inhibit the proliferation, invasion, time-dependent manner, as shown in Fig. 4 and 5.
Fig. 1 Synthesis scheme of target compounds (see Material and Methods section and ESI† section for details). (i) Allyl amine, EtOH, RT; (ii) MeOH,
RT; (iii) zinc dust, trimethylsilyl chloride, THF, RT; (iv) diisopropyl amine, N-butyl lithium, THF; (v) EtOH, reflux; (vi) pyridine, RT.
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Table 1 R-groups of the compounds synthesized and evaluated for
bioactivity against STAT3 in this work
STAT3 phosphorylation, we further functionally analyzed the
inuence of 12g and 12k on the ability of STAT3 to bind DNA in
HCC cells. Analysis of nuclear extracts prepared from HepG2
cells using ELISA-based TransAM™ STAT3 assay kits showed
that 12g inhibited the DNA binding abilities of STAT3 in a dose-
dependent manner (see Fig. 6a). The effect of 12k on STAT3–
DNA binding activity is similar in magnitude but not linearly
related to dose when compared to 12g (Fig. 6b), which may also
be related to stability differences between the thiourea in 12k
and the urea in 12g. These results suggest that 12g can inhibit
the ability of STAT3 to bind DNA in HCC cells signicantly,
giving insight into the mode of action of this compound.
Compounds
R
MW
12a
507.37
12b
12c
12d
12e
545.25
587.43
605.48
623.47
Effect of compound 12g on STAT3 phosphorylation
Given that the presence of phospho-STAT3 at Tyrosine 705 is
known to be a key residue in STAT3 dimerization we further
investigated the ability of 12g to modulate constitutive STAT3
activation in HCC cells via inhibition of phosphorylation at this
residue. For this purpose, HepG2 cells were incubated with 12g
for different time intervals, whole cell extracts were prepared
and the phosphorylation of STAT3 was examined by western
blot analysis using antibodies of STAT3 Tyr705 phosphoryla-
tion. We found that 12g inhibits the constitutive activation of
STAT3 phosphorylation in HepG2 cells in a time-dependent
manner (Fig. 7). Maximum inhibition occurs at 4–6 h, whereas
its effect on the expression of STAT3 protein is negligible.
12f
590.5
Compound 12g suppresses constitutive activation of c-Src
STAT3 has been reported to be activated by soluble tyrosine
kinases of the Src kinase families.5,6 Hence, we determined the
effect of 12g on the constitutive activation of Src kinase in
HepG2 cells and found that 12g suppresses the constitutive
phosphorylation of c-Src kinase in a time-dependent manner
(Fig. 7). The levels of non-phosphorylated Src kinases remained
unchanged under the same conditions.
12g
584.45
12h
652.45
Compound 12g suppresses the constitutive activation of JAK2
Since STAT3 is also activated by soluble tyrosine kinases of the
Janus family (JAKs)5,6 we next determined whether 12g affects
constitutive activation of JAK2 in HepG2 cells. Also, the JAK2/
STAT3 pathway is well-established in HCC carcinoma which
provided additional support for the importance of this partic-
ular experiment. Here we found that 12g suppresses the
constitutive phosphorylation of JAK2 in a time-dependent
manner (see Fig. 7). The levels of non-phosphorylated JAK2
remained unchanged under the same conditions.
12i
12j
606.56
600.51
Effect of compound 12g on prostate cancer (PC3) cells
12k
668.51
In order to further lend support to our hypothesis of compound
12g targeting STAT3 directly, we measured its effect on the
proliferation of PC3 cancer cells which do not phosphorylate
STAT3 constitutively using the MTT method. In agreement with
our hypothesis, we found that 12g had a minimal effect on the
STAT3 DNA binding assay
In order to validate the mode of action of the compounds found proliferation of this cell line (Fig. 8), indicating that the bioac-
to be active in cell proliferation assays and also in affecting tivity of this compound is indeed mediated via STAT3.
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Fig. 2 Cytotoxic effects effect of the compounds described here on MCF-7 cells (plotted as % proliferation inhibition at a concentration of 10
mM), where four of the compounds measured obtained >75% growth inhibition. Standard deviations between the triplicates are indicated. **
indicates p value <0.01; * indicates p value <0.05.
Fig. 3 Cytotoxic effects of PFPs on HepG2 cells (plotted as % proliferation inhibition at a concentration of 10 mM), where two of the compounds
measured obtained >25% growth inhibition. ** indicates p value <0.01; * indicates p value <0.05.
Fig. 4 The effect of 12g on the proliferation of HCC cells is both time- and dose-dependent. HepG2, HUH-7, PLC/PRF/5, and Hep3B cells (5 ꢀ
103 mlꢁ1) were plated in triplicate, treated with indicated concentrations of 12g and then subjected to MTT assay after 24, 48 and 72 hours to
analyze proliferation of cells. Standard deviations between the triplicates are indicated. * indicates p value <0.05.
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Fig. 5 The effect of 12k on the proliferation of HCC cells is both time- and dose-dependent. HepG2, HUH-7, PLC/PRF/5, and Hep3B cells (5 ꢀ
103 ml) were plated in triplicate, treated with indicated concentrations of 12k, and then subjected to MTT assay after 24, 48 and 72 hours to
analyze proliferation of cells. Standard deviations between the triplicates are indicated. * indicates p value <0.05.
Fig. 6 Effect of compound treatment at a concentration of 10 mM on
the ability of STAT3 to bind DNA where both compounds were signifi-
cantly active. HepG2 cells were treated with indicated concentrations of
12k (A) and 12g (B) for 6 h. The nuclear extracts were prepared, and 5 mg
of the nuclear extract protein was used for ELISA based DNA binding
STAT3 constitutively. As it can be seen, much less cytotoxicity
assay as described in “Material and methods”. The results shown are
Fig. 8 Cytotoxic effects of 12g on PC3 cells which do not express
as compared to other cancer cell lines is observed (compared to
Fig. 4 and 5), lending further support that 12g targets the STAT3
representative of three independent experiments, *p < 0.05.
signaling axis.
further target validation hence we next investigated the effect of
12g on CXCL12-induced HCCLM3 cell migration. Using an in
vitro wound healing assay, we found that HCCLM3 cells
migrated faster under the inuence of CXCL12, and this effect
was not observed upon treatment with 12g (Fig. 9). We further
elucidated the effect of compound 12g on CXCL12-induced cell
invasion, a key hallmark of cancer, and it was found that 12g
indeed suppressed CXCL12-induced invasion of HCCLM3 cells
(Fig. 10). Additionally, we also found that the CXCL12 untreated
HCCLM3 cells migrated and invaded more slowly and these
effect were abolished upon treatment with 12g (Fig. 9 and 10),
with all of those observations being consistent of this
Fig. 7 Compound 12g inhibits STAT3 activation in HepG2 cells in a compound targeting the STAT3 axis.
time-dependent manner. Maximum inhibition occurs at 4–6 h,
whereas its effect on the expression of STAT3 protein is negligible.
Molecular modeling of PFPs binding to the SH2 domain of
STAT3
Compound 12g also suppresses the constitutive phosphorylation of c-
Src kinase and JAK2 in a time-dependent manner. The levels of both
non-phosphorylated kinases remained unchanged under the same
conditions. (All data has been obtained at a compound concentration
of 10 mM.)
Molecular modeling was performed to investigate the struc-
tural interaction of the most active STAT3 inhibitors (12g and
12k) with the target in more detail (see Fig. S1 and S2,†
respectively). It appears that two different orientations of the
ligands are possible in the binding site, placing the substitutes
phenyl moieties into different subpockets in each case, and
Effects of compound 12g on HCCLM3 cell migration and
invasion in the presence or absence of CXCL12
It has been reported that STAT3 target gene that promotes that the activity of 12g may be attributed to hydrogen bond/
motility, migration, and invasion of tumor cells,19,20 and for charge interactions between the carbonyl group and the
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Fig. 9 Inhibition of HCC cell migration by 12g, where compound treatment significantly inhibits CXCL12 induced cell migration. Confluent
monolayers of HCCLM3 cells were scarred, and repair was monitored microscopically after 6 h of pre-treatment with compound 12g, before
being exposed to 100 ng mlꢁ1 CXCL12 for 24 h. Width of wound was measured at time zero and 48 h of incubation with and without the
compound. The representative photographs showed the same area at time zero and after 24 h of incubation. Graphs, mean (n ¼ 3); bars, SE. *, P <
0.05. All data has been obtained at a compound concentration of 10 mM.
Fig. 10 A HCC cell invasion assay. It can be seen that 12g significantly reduces CXCL12 induced cell invasion (lower right hand corner of panel A),
compared to untreated cells. B Columns: percentage cell invasion; bars S.E., *p < 0.05. Representative results of two independent experiments
are shown and observations are found to be consistent with 12g targeting the STAT3 signaling axis. All data has been obtained at a compound
concentration of 10 mM.
nitrogen from the tetrahydropyridine ring and Lys591 of fused pyrazoles (‘PFPs’) – that showed antiproliferative effects in
STAT3 SH2 domain.
MCF-7, HepG2, Hep3B and PLC/PRF5 cell lines. One of the most
potent compounds, 12g, inhibited DNA binding of STAT3,
whereas expression itself remains unaltered. In addition this
compound showed complete inhibition of CXCL12-induced cell
Conclusion
In this work we described the synthesis and biological evalua- invasion and associated wound healing. 12g failed to inhibit the
tion of a novel class of inhibitors of STAT3 signaling – pyridine- proliferation of PC3 prostate cancer cells, a cell line which does
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not have constitutively phosphorylated STAT3, which corrobo- tetramethylsilane (TMS) as internal standard. Mass and purity
rates our hypothesis of 12g inhibition of the STAT3 signaling were recorded on a LC-MSD-Trap-XCT.
pathway. HCCLM3 cell migration and invasion however are
inhibited by this compound, in line with the previously reported
involvement of STAT3 in cell motility. Molecular modeling
Biology
studies suggest different binding modes of 12g and 12k, giving a
Biological reagents. MTT, Tris, glycine, NaCl, SDS, and BSA,
possible explanation for the different effects observed experi- were purchased from Sigma-Aldrich (St. Louis, MO). Dulbecco’s
mentally. Given the importance of STAT3 modulation in various Modied Eagle Medium (DMEM), fetal bovine serum (FBS),
cancers the results presented here hence warrant further study 0.4% trypan blue vital stain, and antibiotic–antimycotic mixture
of those compounds in more complex systems.
were obtained from Invitrogen. Rabbit polyclonal antibodies to
STAT3 and mouse monoclonal antibodies against phospho-
STAT3 (Tyr 705) were obtained from Santa Cruz Biotechnology
(Santa Cruz, CA). Antibodies to phospho-specic Src (Tyr416),
Src, phospho-specic JAK2 (Tyr1007/1008) and JAK2 were
purchased from Cell Signalling Technology (Beverly, MA).
CXCL12 was purchased from ProSpec-Tany TechnoGene Ltd.
(Rehovot, Israel). Goat anti-rabbit-horse radish peroxidase
(HRP) conjugate and goat anti-mouse HRP were purchased
from Sigma-Aldrich (St Louis, MO).
Cell lines. Human hepatocellular carcinoma (HCC) cell lines
(HepG2, Hep3B and PLC/PRF5), and prostate cancer PC3 cells
were obtained from American Type Culture Collection (Mana-
ssass, VA). HUH-7 and HCCLM3 cells were kindly provided by
Prof. Kam Man Hui, National Cancer Centre, Singapore. All the
ve HCC cell lines were cultured in DMEM containing 1ꢀ
antibiotic–antimycotic solution with 10% FBS. PC3 cells were
cultured in Roswell Park Memorial Institute-1640 (RPMI-1640)
medium containing 1ꢀ antibiotic–antimycotic solution with
10% FBS.
Materials and methods
Target site and scaffold choice
In order to narrow down chemical space for the synthesis of
STAT3 inhibitors we used both the crystal structure of the
recently published STAT3b homodimer (PDBID: 1BG1)21 as well
as a number of heterocycles known to target this protein as a
starting point, such as the pyrazole moiety in Celecoxib, the
benzimidazole in Dovitinib, the indazole in ABT-869 and the
purine ring.22–26 Our attempt was here to emulate similar
ligand–protein interactions with the SH2 domain of STAT3 as
suggested in a previous study for cryptotanshinone27 and to
hence target the polar/basic region around Lys591, Arg609,
Glu612 and Ser613 (including p-Tyr705, which is important for
the dimerisation event following phosphorylation), the hydro-
phobic region around Ile597, Trp623, Ile634 and Val637, as well
as the charged amino acids (surrounded by hydrophobic resi-
dues) Glu594, Glu592 and Arg595. The manual design process
led us conclude that the pyridine-fused pyrazole (‘PFP’) scaffold
was suitable both from an activity perspective, as well as not
bearing any obvious liabilities from the medicinal chemistry
perspective.
DNA binding assay. DNA binding was performed using a
STAT3 DNA binding ELISA kit (Active Motif, Carlsbad, CA, USA).
Briey, nuclear extracts (5 mg) from compound treated cells were
incubated in a 96-well plate coated with oligonucleotide con-
taining the STAT3 specic DNA probe. Bound STAT3 was then
detected using a specic primary antibody. An HRP-conjugated
secondary antibody was then applied to detect the bound
Synthesis
The synthesis of PFPs involved six steps via Reformatsky reac- primary antibody and provided the basis for colorimetric
tion, Dickman cyclysation, and cyclocondensation of 2,4-phenyl quantication. The enzymatic product was measured at 450 nm
hydrazine (9) with cyano ketone (8) (Fig. 1). Finally, compound with a microplate reader (Tecan Systems, San Jose, CA, USA).
10 was derivatised using various acid chlorides, sulphonyl
MTT assay. The antiproliferative effect of compounds
chloride, isocyanates, and thiocyanates in the presence of against tumor cells was determined by the MTT dye uptake
pyridine to give the corresponding title compounds such as method as described previously.18 Briey, the cells (5 ꢀ
tetrahydroindazole carboxymides 12(a–c), sulfonamides 12(d– 103 mlꢁ1) were incubated in triplicate in a 96-well plate in the
e), urea derivatives 12(f–h) and thiourea derivatives 12(i–k). presence or absence of indicated concentration of compounds
Characterization was performed via IR, 1H NMR, 13C NMR, and in a nal volume of 0.2 ml for different time intervals at 37 ꢂC.
Elemental Analysis. For individual compound syntheses see Thereaer, 20 ml MTT solution (5 mg mlꢁ1 in PBS) was added to
ESI† section.
each well. Aer a 2 h incubation at 37 ꢂC, 0.1 ml lysis buffer
Analytical chemistry. All analytical thin layer chromatog- (20% SDS, 50% dimethylformamide) was added; incubation
raphy was performed with E. Merck silica gel 60F254 aluminum was done for 1 h at 37 ꢂC and the optical density (OD) at 570 nm
sheets and was visualized with UV light. The following mobile was measured by Tecan plate reader.
phases were employed for TLC: chloroform, methanol and
Western blotting. For detection of various proteins,
hexane, ethyl acetate in different ratios. The instrumental compound 12g-treated whole-cell extracts were lysed in lysis
techniques employed for the characterization of the newly buffer (20 mM Tris (pH 7.4), 250 mM NaCl, 2 mM EDTA (pH
synthesized compounds includes IR NMR and mass spectros- 8.0), 0.1% Triton X-100, 0.01 mg mlꢁ1 aprotinin, 0.005 mg mlꢁ1
copy. IR spectra were recorded on an FT-IR spectrometer. 1H leupeptin, 0.4 mM PMSF, and 4 mM NaVO4). Lysates were spun
NMR spectra were recorded on CDCl3, DMSO-d6 solution at 14 000 rpm for 10 min to remove insoluble material and
using a 400 and 300 MHz Fourier Transform NMR with resolved on a 7.5% SDS gel. Aer electrophoresis, the proteins
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were electrotransferred to a nitrocellulose membrane, blocked of Syngene International limited, India for valuable
with 5% non-fat milk, and probed with various antibodies suggestions.
(1 : 1000) overnight at 4 ꢂC. The blot was washed, exposed to
HRP-conjugated secondary antibodies for 1 h, and nally
examined by chemiluminescence (ECL; GE Healthcare, Little
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Acknowledgements
This research was supported by the Singapore Ministry of 19 A. Subramaniam, M. K. Shanmugam, E. Perumal, F. Li,
Health’s National Medical Research Council to GS under its IRG
funding scheme, University Grants Commission to Basappa (41-
257-2012-SR) and Bhadregowda (41-316-2012-SR), Vision Group
A. Nachiyappan, X. Dai, S. N. Swamy, K. S. Ahn,
A. P. Kumar, B. K. Tan, K. M. Hui and G. Sethi, Biochim.
Biophys. Acta, 2013, 1835, 46–60.
Science and Technology to Basappa, Government of Karnataka, 20 P. Rajendran, T. H. Ong, L. Chen, F. Li, M. K. Shanmugam,
INDIA, and Department of Science and Technology (no. SR/FT/
LS-142/2012) to Basappa. AB thanks Unilever for funding. We
thank Dr P. Sathyashankar, and Dr Subhendu Kumar Mohanty
S. Vali, T. Abbasi, S. Kapoor, A. Sharma, A. P. Kumar,
K. M. Hui and G. Sethi, Clin. Cancer Res., 2011, 17, 1425–
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