Quinoides and VEGFR2 TKIs influence the fate of hepatocellular carcinoma and its cancer stem cells

Article abstract of DOI:10.1039/c6md00392c

Bioactivities of quinoides 1-5 and VEGFR2 TKIs 6-10 in hepatocellular cancer (HCC) and cancer stem cells (HCSCs) were studied. The compounds exhibited IC₅₀ values in μM concentrations in HCC cells. Quinoide 3 was able to eradicate cancer stem cells, similar to the action of the stem cell inhibitor DAPT. However, the more cytotoxic VEFGR TKIs (IC₅₀: 0.4-3.0 μM) including sorafenib, which is the only FDA approved drug for the treatment of HCC, enriched the hepatocellular cancer stem cell population by 2-3 fold after treatment. An aggressiveness factor (A_F) was proposed to quantify the characteristics of drug candidates for their ability to eradicate the CSC subpopulation. Considering the tumour heterogeneity and marker positive cancer stem cell like subpopulation enrichment upon treatments in patients, this study emphasises the importance of the chemotherapeutic agent choice acting differentially on all the subpopulations including marker-positive CSCs.

Full text of DOI:10.1039/c6md00392c

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RESEARCH ARTICLE
Quinoides and VEGFR2 TKIs influence the fate of
hepatocellular carcinoma and its cancer stem
cells†‡
Cite this: DOI: 10.1039/c6md00392c
Deniz Cansen Kahraman,a Gilles Hanquet,b Loïc Jeanmart,c Steve Lanners,c
de
f
Peter Šramel,de Andrej Boháč
and Rengul Cetin-Atalay
*
*
Bioactivities of quinoides 1–5 and VEGFR2 TKIs 6–10 in hepatocellular cancer (HCC) and cancer stem cells
(HCSCs) were studied. The compounds exhibited IC50 values in μM concentrations in HCC cells. Quinoide
3 was able to eradicate cancer stem cells, similar to the action of the stem cell inhibitor DAPT. However,
the more cytotoxic VEFGR TKIs (IC50: 0.4–3.0 μM) including sorafenib, which is the only FDA approved drug
for the treatment of HCC, enriched the hepatocellular cancer stem cell population by 2–3 fold after treat-
ment. An aggressiveness factor (AF) was proposed to quantify the characteristics of drug candidates for
their ability to eradicate the CSC subpopulation. Considering the tumour heterogeneity and marker positive
cancer stem cell like subpopulation enrichment upon treatments in patients, this study emphasises the im-
portance of the chemotherapeutic agent choice acting differentially on all the subpopulations including
marker-positive CSCs.
Received 15th July 2016,
Accepted 27th September 2016
DOI: 10.1039/c6md00392c
www.rsc.org/medchemcomm
cells and acquire resistance to chemotherapy in most of the
cancer types.3,4 Hence, it is crucial to develop novel drugs
Introduction
Hepatocellular carcinoma (HCC) is the sixth most frequent
and second most deadly cancer worldwide.1 HCC patients are
resistant to chemotherapy and radiotherapy, because conven-
tional therapies can only reduce the bulk of the tumour mass
but are unable to restrain tumour regrowth and relapse. HCC
is a highly heterogeneous tumour in terms of morphology
and clinical outcome.2 The enrichment of cancer stem cells is
one of the main reasons behind failure in treatment of HCC
patients. Sorafenib (6), a multi-kinase inhibitor acting on
VEGFR2-TK and also the only FDA approved drug for the
treatment of HCC patients, inhibits proliferation and migra-
tion of tumour cells and angiogenesis. Recently, it has been
reported that many patients develop resistance to sorafenib.
This was explained by the enrichment of cancer stem cells
that have the capacity to self-renew, differentiate into cancer
against the differentiated cancer cells as well as liver cancer
stem cells in order to successfully eradicate liver cancer. Liver
cancer stem cells could be identified and isolated by several
surface markers including CD133, CD90, CD44, CD13,
EpCAM, OV-6, CD24, DLK1 and ICAM-1.5 Cells that carry one
or two of these markers were shown to possess CSC features.
The epithelial–mesenchymal transition (EMT) is a critical
step for stemness. In this study, we have selected liver cancer
cell lines with different phenotypic properties based on their
genotypes. Huh7 and Hep3B cells are known to have epithe-
lial features (well-differentiated), whereas Mahlavu and SNU-
475 cells were characterized as mesenchymal-like (poorly-dif-
ferentiated) cells.6 Huh7 and Hep3B cells express genes asso-
ciated with epithelial like properties. These cells express the
HCC marker AFP along with the epithelial marker E-
cadherin, while Mahlavu and SNU-475 cells don't express AFP
and E-cadherin genes while they express high levels of
vimentin protein.6 Furthermore, Huh7 and Hep3B cells have
normal migratory properties, but Mahlavu and SNU-475 cells
have high migratory properties which may be due to their dif-
ferential PI3K/Akt pathway activities. It was shown that
Mahlavu and SNU-475 cells have constitutively active Akt pro-
tein due to loss of the PTEN tumor suppressor.7 Current
studies and clinical trials focus on the anti-CSC compounds
targeting extracellular mediators or cell surface molecules as
well as molecules involved in EMT and metastasis.8 Thus, it
is crucial to define which marker is efficient to detect and
a Dept. of Mol. Biol. and Gen., Bilkent University, Ankara, 06800, Turkey
b Laboratoire Syncat, UMR CNRS 7509, ECPM, Université de Strasbourg, 25 rue
Becquerel, 67087 Strasbourg, France
c Department of Chemistry and Namur Medicine & Drug Innovation Center
(NAMEDIC), University of Namur, 61 rue de Bruxelles, 5000 Namur, Belgium
d Faculty of Natural Sciences, Comenius University, Ilkovičova 6,
Mlynskádolina842 15 Bratislava (PŠ, AB), Slovakia
e Biomagi Ltd., Mamateyova 26, 851 04 Bratislava, Slovakia
f Cancer Systems Biology Laboratory, Graduate School of Informatics, ODTU,
Ankara, 06800, Turkey. E-mail: rengul@metu.edu.tr
† The authors declare no competing interests.
‡ Electronic supplementary information (ESI) available. See DOI: 10.1039/
c6md00392c
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Table 2 IC50 values and AF factors of quinoides 1–5 and VEGFR2-TKIs
6–10 in (A) Huh7 and Hep3B epithelial cells and of quinoides 1–5 in (B)
Mahlavu (MV) and SNU-475 (S-475) mesenchymal cells
Fig. 1 The structures of tested quinoides 1–5.
analyse cancer stem cell markers in each cell line, which we
studied by flow cytometry.
Quinones and quinone-like compounds seem to be prom-
ising candidates against cancer stem cells; however, they are
mainly organotoxic. It has been reported that minor changes
in the side chains of the quinone structure can lead to a
strong variation in the biological toxicity;9–11 thus,
modelization of various quinoid derivatives with strong in-
hibitory activity and low toxicity is conceivable. Therefore, we
prepared a series of quinoides, 1–5 (Fig. 1), and evaluated
their activities in different HCC cell lines and their cancer
stem cell (HCSC) population (Tables 1, 2A and B and Fig. 5).
Furthermore, VEGFR2 TKIs 6–10 (Fig. 2) were also screened
in HCC cell lines (Tables 1 and 2A). Compound 6 is sorafenib
tosylate (Nexavar®), and compound 9 was developed by
GlaxoSmithKline as a drug candidate to inhibit VEGFR2 TK.
Compounds 7, 8 and 10 were designed by Biomagi, Ltd. to
modulate VEGFR2 TK activity.
Results and discussion
Chemistry
Fig. 2 VEGFR2 TKIs 6–10 used in this study and their origin and
VEGFR2 IC50 activities that were obtained through
service in Germany (www.proqinase.com).
a commercial
Compounds 1, 2a and 2b were prepared according to our pre-
vious
work
starting
from
commercially
available
2-methylhydroquinone (S1) (Scheme 1).12
Compounds 3 and 5 were prepared (Scheme 2) according
to Carreño's procedure with slight modifications (see the
ESI‡).13–15
The new quinoide 4 was obtained from vanillin (S7) in
very good overall yield using the sequence depicted in
Scheme 3.
VEGFR2 TKI 6 was obtained from Bayer Inc. Synthesis of 7
was described by Lintnerová et al.16 Compound 9 was pre-
pared according the procedure described by Harris et al.17
Novel compounds 8 and 10 were synthesized using the se-
quence depicted in Scheme 4 (see also the ESI‡).
Table 1 Screening of quinoides 1–5 and VEGFR2-TKIs 6–10 in HCC cell
lines
IC50 (μM)a
Compound
Huh7
Hep3B
Mahlavu
1
2a
2b
3
4
5
7.0 0.8
8.0 0.9
5.5 0.4
20.0 1.7
10.6 2.0
Ni
12.0 1.7
5.5 1.8
4.3 0.7
17.4 2.1
9.9 2.3
Ni
18.3 0.8
14.5 1.3
11.6 0.2
31.3 0.3
11.3 0.6
Ni
Biological evaluation
Bioactivities of quinoides and VEGFR2-TKs in HCC cells.
NCI-SRB assay was initially performed to define the inhibi-
tory concentrations (IC50 values) of compounds in HCC cell
lines Huh7, Hep3B, and Mahlavu. Results have shown that al-
most all compounds (except for 5) had IC50 values in μM con-
centrations for all cell lines (Table 1). DAPT (γ-secretase in-
hibitor) was used as a control for CSC inhibition.
6
7
8
9
1.6 0.8
2.8 1.3
1.7 0.7
1.5 0.7
0.4 0.3
5.0 1.2
1.5 0.3
2.0 0.9
2.1 0.3
0.7 0.1
0.9 0.3
5.0 2.0
0.7 0.4
3.0 0.5
0.8 0.5
1.2 0.4
1.1 0.7
5.0 1.5
10
DAPT
Expression of cancer stem cell markers in HCC cell lines.
CSC marker expression varies among cell lines of HCC, since
a
Experiments were performed in triplicate; Ni: no growth inhibition.
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Scheme
1 Preparation of quinones 1, 2a and 2b from
2-methylhydroquinone (S1).
Scheme 4 Syntheses of 8 and 10.
fore, the expression of mutant p53 causes differential activa-
tion of the β-catenin protein and its downstream mesenchy-
mal proteins such as E-cadherin, vimentin, snail and slug.
The significantly higher expression of β-catenin20 due to the
lack of p53 protein in Hep3B cells correlates with the higher
expression of EpCAM and CD133 markers in this cell (Fig. 3).
Effects of quinoides and VEGFR2-TKIs on hepatocellular
cancer stem cells (HCSCs). It is essential to discover novel
compounds that were able to alter the enrichment of the CSC
Scheme
dimethoxybenzene.
2
Syntheses of quinones
3
and
5
from 1,4-
Scheme 3 Synthesis of compound 4.
liver CSCs exist heterogeneously and these features determine
the characteristics of the cancer (tumourigenicity and meta-
static tendency). It has previously been reported that the epi-
thelial cell adhesion molecule (EpCAM) and CD133 express-
ing cells are more likely to execute epithelial features,
whereas CD90 expressing cells are more likely to be mesen-
chymal.18,19 Expression of these markers in HCC cell lines
was analyzed using flow cytometry. It was shown that CSCs of
Huh7 cells could be identified by CD133 and EpCAM positiv-
ity (20–30%). CSCs of Mahlavu and SNU-475 could be identi-
fied by CD90 positivity (0.2–0.6%) (Fig. 3).
As stated above, the EMT is critical for cancer cell
stemness. Huh7 and Hep3B cells carry epithelial features,
whereas Mahlavu and SNU-475 cells carry migratory
mesenchymal-like properties.6,7 Furthermore, Huh7 and
Hep3B cells differ in their p53 gene status: Huh7 cells express
p53IJY220S), but Hep3B cells are null for the TP53 gene. There-
Fig.
3 Flow cytometry analysis of HCC cell lines indicating the
positivity of HCC cell lines for cancer stem cell markers CD133,
EpCAM and CD90. Top row: IgG isotype controls, middle row: cells
stained for CD133 and EpCAM markers, bottom row: cells stained for
CD90.
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population as well as the cancer cells. For this reason, com-
pounds 1–10 that were initially tested against HCC cell lines
were further studied to determine the changes in the CSC
marker positivity of Huh7 cells. Cells that were killed by the
treatment were discarded, and the remaining cells were used
in flow cytometry analyses. Interestingly, some of the com-
pounds were able to reduce the percentage of CSC popula-
tion, while some had the adverse effect on Huh7 cells that
enriched the CSC subpopulation (Fig. 4). It was observed that
unlike VEGFR2-TKIs, some of the quinoides were able to at-
tenuate the CSC side population in Huh7 cells.
cell inhibitor, DAPT (Fig. 5). Altogether, the results have
shown that compound 3 is potentially capable of impairing
the cancer stem cells in HCC cells with a mechanism not yet
discovered.
On the other hand, treatments of cells with VEGFR2-TKIs
6–10 were shown to enrich the cancer stem cell population of
Huh7 and Hep3B cells at about 1.7–2.9 times as opposed to
the effect of DAPT (Fig. 4 and Table 2A).
Conclusions
Due to the distinct dispersion of CD90 cells, we decided to
include SNU-475 cells for testing the anti-HCSC activities of
quinoides. Therefore, we also determined the bioactivities of
quinoides in SNU475 cells. The IC50 values were 5.5 1.4, 10.5
1.5, 6.1 2.0, 0.3 1.2, 9.0 2.1, and 0.6 1.1 μM for com-
pounds 1, 2a, 2b, 3, 4, and DAPT, respectively. No growth inhi-
bition was observed for compound 5 (Fig. 5 and Table 2B).
Although compounds 6–10 were stronger HCC inhibiting
agents as quinoides 1–5, they caused enrichment of Huh7
HCSCs in the rest of the HCC after treatment. Therefore, the
positive cytotoxic effect of 6–10 was discredited by their abil-
ity to leave the HCC residue fraction after treatment enriched
with aggressive HCSC cells that are highly susceptible to dis-
ease relapse and that acquired drug resistance frequently ob-
served after HCC treatment in clinics.
There is only one literature report describing macrocyclic
benzoquinone herbimycin A [CAS: 70563-58-5, BRN: 4834067]
that inhibits (61–78%) human bone marrow mesenchymal
stem cells at 1–5 μM concentration.21 We present here for the
first time the screening of benzoquinone compounds in
CSCs. One of the achievements of our pivotal study is identi-
fication of quinoide 3 as an exceptional compound to treat
HCSCs. Although its mechanism is not yet known, we hy-
pothesize different behaviours of screened quinoides 1–5
based on their structural differences, benzoquinone reactivity
and general medicinal chemistry knowledge. All quinoides
1–5 possess a chemically reactive benzoquinone fragment
that can likely be an irreversible inhibitor binding to a bio-
logical target e.g. by the Michael reaction with a cysteine
amino acid residue. The most active quinoide 3 in Mahlavu
and SNU-475 is the one that misses most of substitutions on
its benzoquinone skeleton compared to the other tested
quinoides. The steric and electronic properties of 3 could be
a reason why quinoide 3 is the most active one included
(Fig. 5). In the case of the SNU-475 tumour, there are three
active quinoides (1, 2a and 3); therefore, other mechanismIJs)
can also be included. As could be seen, all VEGFR2 TKIs 6–10
are cytotoxic to HCC cell lines (IC50 = 3.0–0.4 μM, Table 1),
and at the same time, they are less toxic towards HCSCs since
it was observed that cancer stem cells were enriched up to 3
times after treatment (Fig. 5 and Table 2A). The most syner-
gic case would be to identify cytotoxic compounds for both
HCCs and HCSCs. The results obtained by screening of
VEGFR2 TKIs can deliver two positive consequences: i)
VEGFR2 TKIs can be used to enrich the % of stem cells in
the final fraction before stem cell isolation for research pur-
poses. Moreover, such stem cells can remember VEGFR2 TKI
treatment and can be used for the development of the next
generation of inhibitors. ii) It is obvious that experimental
compounds can be differently cytotoxic towards heteroge-
neous tumour cells. Their different influence on CSCs should
be quantified separately because of its practical meaning.
In cancer therapeutics, it is important to identify treat-
ment regimens against cancer stem cells as well as tumor
cells, because patients suffer from relapse or incomplete re-
covery after treatment if it fails to eliminate CSCs. The per-
centage of DMSO treated cells in flow cytometry analysis rep-
resents the initial CSC marker positivity of the cells before
treatment (Fig. 4) Thus, we suggest that once the cells were
treated with a compound, the number of cells with marker
Further analyses were carried out on Hep3B, Mahlavu and
SNU-475 cells to test whether the same effect could be ob-
served in other HCC cell lines as well. Among all compounds
tested, 3 was able to attenuate the CSCs especially in
mesenchymal-like cells, surpassing the efficacy of the stem
Fig. 4 Expression of stem cell markers CD133 and EpCAM in Huh7
cells treated with (A) quinoides 1–5 and (B) VEGFR2-TKIs 6–10 as
shown by flow cytometry analysis.
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Fig. 5 Flow cytometry analysis of HCC cell lines after 72 hours of treatment with quinoides 1–5. (A) Huh7, (B) Hep3B, (C) Mahlavu, and (D) SNU-
475. Cancer stem cells were defined by their positivity for CSC markers, CD133 together with EpCAM, or CD90. Each treatment was compared to
its corresponding DMSO control to define the changes in percentage of double positive population. DAPT is used as positive control for CSC
inhibition. The bar graphs indicate the fold change in positivity for markers between DMSO control and experimental groups.
positivity can be compared with the DMSO control in order
to define the fold change in the percentage of cancer stem
cells. Since only the cells that stay alive after treatment are
analyzed (see methods), it is important to emphasize the den-
sity of the cancer stem cells before and after treatment.
Therefore, the compounds that cause a decrease or enrich-
ment in cancer stem cell population can be quantified simply
by normalizing the treated cell population by DMSO controls.
Here, we suggest the aggressiveness factor (AF), a new
characteristic quantifying the risk for certain compounds to
be able to develop a more aggressive disease: AF (exp. cmpd.)
= [(total number of CSCs after exp. cmpd. treatment)/(total
number of CSCs before treatment)].
The low AF value is an indication for the quality of a drug
candidate toward cancer cells. Such molecules don't possess
drug resistance or induce more aggressive tumours (Table 2). AF
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reflects the fold increase of CSCs that survived the compound
treatment independently of the shrinkage of the tumour itself.
The compounds with AF values below 1 can be regarded as
molecules which reduce the cancer stem cell population,
whereas AF values above 1 signify molecules that enrich the
cancer stem cell population. Thus, the usage of AF values de-
fines the success or failure of the compounds in affecting
cancer cells toward cancer stem cell population.
NCI-60 sulforhodamine B (SRB) cytotoxicity assay
Huh7 and Hep3B (2000 cells per well), SNU-475 (1000 cells
per well) and Mahlavu (1000 cells per well) cells were inocu-
lated into 96-well plates (150 μl per well). After 24 hours, mol-
ecules of interest and DMSO control were applied in concen-
trations of 40 μM to 2.5 μM in serial dilutions. After 72 h of
treatment, cells were fixed with cold 10% (w/v) trichloroacetic
acid (MERCK) for an hour. Then, the wells were washed with
ddH2O and dried. 50 μl of 0.4% SRB dye (Sigma-Aldrich) was
applied to each well and incubated at room temperature for
10 min. Each well was washed with 1% acetic acid and left
for air-drying. SRB dye was solubilised using 100 μl per well
10 mM Tris-Base solution, and the absorbance was measured
at 515 nm. The experiment was performed in triplicate, and
the absorbance values were normalized to DMSO controls.
Cancer stem cell-like subpopulations carry behaviors such
as higher tumor-forming and metastasis capacities along
with resistance to antitumor drugs which allows tumors to
survive and relapse.8 The analysis of the action of the chemo-
therapeutics that deplete the CSC-like population by the fol-
lowing surface markers may enumerate the qualities of the
compounds and may allow one to assess their differential ac-
tion. Our results represent parallel findings with sorafenib
(6) and other compounds and allow comparative analysis of
the compounds toward cancer stem cell marker positive cell
populations. The AF concept introduced in this study clearly
demonstrates the differential action of the compounds.
It is known that patients used to develop resistance to-
wards sorafenib (6). This is consistent with our data with
sorafenib (6) having an AF value that is 2 fold higher com-
pared to that of the DMSO control while maintaining good
cytotoxicity through the low IC50 values (Table 2A). However,
the known CSC inhibitor (DAPT) has low AF values and its
IC50 is much higher than that for sorafenib (6). Furthermore,
while quinoide 5 does not possess cytotoxic actions (for each
HCC line) and 3 has high IC50 values against Huh7, Hep3B
or MV (Mahlavu) cells, they both significantly reduce the CSC
marker positive subpopulations in mesenchymal Mahlavu
and SNU-475 cells (Tables 2A and B).
Flow cytometry
HCC cells are inoculated into 100 mm2 culture dishes (100
000–200 000 cells). 24 hours later, cells were treated with the
compounds (IC100 conc.) for 72 hours. Dead cells that no lon-
ger remained attached to the surface of the culture plates
were discarded through vacuum aspiration and cells that
remained attached were collected to be fixed with 4% para-
formaldehyde for 30 minutes. Huh7 and Hep3B cells were
stained for cancer stem cell markers using anti-CD133/1
(AC133)–biotin (Miltenyi, 130-090-664), anti-biotin–PE
(Miltenyi, 130-090-756), and anti-EpCAM–FITC (Miltenyi, 130-
080-301), whereas Mahlavu and SNU-475 cells were stained
using anti-CD90–FITC (Miltenyi, 130-095-403). For isotype
controls, mouse IgG1 isotype control–FITC conjugate
(Miltenyi, 130-092-213) and mouse IgG1 isotype control anti-
body–biotin conjugate (Miltenyi, 130-093-018) were used.
Staining of cells was performed according to the manufac-
turer's protocol. Cells were analyzed using the BD Accuri C6
Flow Cytometer and Software (BD Biosciences). The same
staining procedure was applied for the analysis of HCC cells
in order to determine the CSC marker positivity.
The normalized aggressiveness factor allows selection of
promising experimental compounds possessing lower proba-
bility to form aggressive tumours in comparison to a particu-
lar drug with known clinical behaviour. The methodology can
serve as a simple and valuable tool for pre-clinical screening.
Experimental section
Syntheses of compounds 1–10
Acknowledgements
Liver cancer stem cell studies and DCK supported by
TUBITAK grant #113S540. COST Actions: CM1106, CA15135,
VEGA 1/0634/13, APV SK-FR-2015-0014 and Biomagi, Ltd. are
acknowledged for chemistry. Sorafenib tosylate (6) was
obtained as a gift from Bayer, Inc.
The syntheses and physicochemical properties of prepared
organic compounds 1–10 can be found in the ESI.‡
Cell culture
Huh7 and Mahlavu, human hepatocellular carcinoma (HCC)
cell lines, were maintained in Dulbecco's modified Eagle's
medium (DMEM) (Invitrogen/GIBCO), supplemented with
10% fetal bovine serum (FBS) (Invitrogen/GIBCO) and 0.1
mM nonessential amino acid, whereas SNU-475 cells were
maintained in RPMI (Invitrogen/GIBCO), supplemented 10%
fetal bovine serum (FBS) and 2 mM L-glutamine. Both media
contained 100 units per mL penicillin and 100 units per mL
streptomycin. Cells were grown at 37 °C in a humidified incu-
bator under 5% CO2.
References
1 B. W. Stewart and C. P. Wild, World Cancer Report 2014, 2014.
2 T. Yamashita and X. W. Wang, J. Clin. Invest., 2013, 123(5),
1911–1918.
3 H.-W. Xin, C. M. Ambe, D. M. Hari, G. W. Wiegand, T. C.
Miller, J. Q. Chen, A. J. Anderson, S. Ray, J. E. Mullinax, T.
Koizumi, R. C. Langan, D. Burka, M. A. Herrmann, P. K.
Goldsmith, A. Stojadinovic, U. Rudloff, S. S. Thorgeirsson
and I. Avital, Gut, 2013, 62, 1777–1786.
Med. Chem. Commun.
This journal is © The Royal Society of Chemistry 2016

View Article Online
MedChemComm
Research Article
4 A. K.-M. Chow, L. Ng, C. S.-C. Lam, S. K.-M. Wong, T. M.-H.
Wan, N. S.-M. Cheng, T. C.-C. Yau, R. T.-P. Poon and
R. W.-C. Pang, PLoS One, 2013, 8, e78675.
5 J.-H. Sun, Q. Luo, L.-L. Liu and G.-B. Song, World J.
Gastroenterol., 2016, 22, 3547–3557.
6 H. Yuzugullu, K. Benhaj, N. Ozturk, S. Senturk, E. Celik, A.
Toylu, N. Tasdemir, M. Yilmaz, E. Erdal, K. C. Akcali, N.
Atabey and M. Ozturk, Mol. Cancer Ther., 2009, 8, 90.
7 I. Durmaz, E. B. Guven, T. Ersahin, M. Ozturk, I. Calis and
R. Cetin-Atalay, Phytomedicine, 2016, 23, 42–51.
8 F. Marcucci, C. Rumio and F. Lefoulon, Front. Radiat. Oncol.,
2016, 6, 115.
9 T. J. Monks, R. P. Hanzlik, G. M. Cohen, D. Ross and D. G.
Graham, Toxicol. Appl. Pharmacol., 1992, 112, 2–16.
10 T. J. Monks and D. C. Jones, Curr. Drug Metab., 2002, 3, 425–438.
11 K. Ollinger and A. Brunmark, J. Biolumin. Chemilumin.,
1991, 266, 21496–21503.
14 M. C. Carreño, J. L. G. Ruano, M. A. Toledo and A. Urbano,
Tetrahedron Lett., 1994, 35, 9759–9762.
15 M. A. Brimble, L. J. Duncalf and D. C. W. Reid, Tetrahedron:
Asymmetry, 1995, 6, 263–269.
16 L. Lintnerová, M. García-Caballero, F. Gregáň, M.
Melicherčík, A. R. Quesada, J. Dobiaš, J. Lác, M. Sališová and
A. Boháč, Eur. J. Med. Chem., 2014, 72, 146–159.
17 P. A. Harris, M. Cheung, R. N. Hunter, M. L. Brown, J. M.
Veal, R. T. Nolte, L. Wang, W. Liu, R. M. Crosby, J. H.
Johnson, A. H. Epperly, R. Kumar, D. K. Luttrell and J. A.
Stafford, J. Med. Chem., 2005, 48, 1610–1619.
18 T. Yamashita and S. Kaneko, J. Gastroenterol., 2014, 49,
1105–1110.
19 Z. F. Yang, D. W. Ho, M. N. Ng, C. K. Lau, W. C. Yu, P. Ngai,
P. W. K. Chu, C. T. Lam, R. T. P. Poon and S. T. Fan, Cancer
Cell, 2008, 13, 153–166.
20 T. Cagatay and M. Ozturk, Oncogene, 2002, 21,
7971–7980.
21 S. Mruthyunjaya, R. Manchanda, R. Godbole, R. Pujari, A.
Shiras and P. Shastry, Biochem. Biophys. Res. Commun.,
2010, 391, 43–48.
12 D. A. Lanfranchi and G. Hanquet, J. Organomet. Chem.,
2006, 71, 4854–4861.
13 M. C. Carreño and J. L. García Ruano, Synthesis, 1992, 1992,
651–653.
This journal is © The Royal Society of Chemistry 2016
Med. Chem. Commun.