Design, synthesis and biological evaluation of a new thieno[2,3-d]pyrimidine-based urea derivative with potential antitumor activity against tamoxifen sensitive and resistant breast cancer cell lines

Article abstract of DOI:10.1080/14756366.2020.1804383

Breast cancer (BC) and endocrine resistance to chemotherapy are challenging problems where angiogenesis plays fundamental roles. Thus, targeting of VEGFR-2 signalling pathway has been an attractive approach. In this study, we synthesised a new sorafenib a

Full text of DOI:10.1080/14756366.2020.1804383

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Design, synthesis and biological evaluation
of a new thieno[2,3-d]pyrimidine-based urea
derivative with potential antitumor activity
against tamoxifen sensitive and resistant breast
cancer cell lines
Marwa Sharaky , Marwa Kamel , Marwa A. Aziz , Mervat Omran , Monira M.
Rageh , Khaled A. M. Abouzid & Samia A. Shouman
To cite this article: Marwa Sharaky , Marwa Kamel , Marwa A. Aziz , Mervat Omran , Monira
M. Rageh , Khaled A. M. Abouzid & Samia A. Shouman (2020) Design, synthesis and biological
evaluation of a new thieno[2,3-d]pyrimidine-based urea derivative with potential antitumor activity
against tamoxifen sensitive and resistant breast cancer cell lines, Journal of Enzyme Inhibition and
Medicinal Chemistry, 35:1, 1641-1656, DOI: 10.1080/14756366.2020.1804383
To link to this article: https://doi.org/10.1080/14756366.2020.1804383
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2020, VOL. 35, NO. 1, 1641–1656
https://doi.org/10.1080/14756366.2020.1804383
RESEARCH PAPER
Design, synthesis and biological evaluation of a new thieno[2,3-d]pyrimidine-
based urea derivative with potential antitumor activity against tamoxifen sensitive
and resistant breast cancer cell lines
Marwa Sharakya, Marwa Kamela, Marwa A. Azizb, Mervat Omrana, Monira M. Ragehc, Khaled A. M. Abouzidb,d and
Samia A. Shoumana
b
aDepartment of Cancer Biology, Pharmacology Unit, National Cancer Institute, Cairo University, Cairo, Egypt; Department of Pharmaceutical
c
Chemistry, Faculty of Pharmacy, Ain Shams University, Abbassia, Cairo, Egypt; Department of Biophysics, Faculty of Science, Cairo University,
d
Giza, Egypt; Department of Organic and Medicinal Chemistry, Faculty of Pharmacy, University of Sadat City, Menoufia, Egypt
ABSTRACT
ARTICLE HISTORY
Received 27 January 2020
Revised 22 July 2020
Accepted 23 July 2020
Breast cancer (BC) and endocrine resistance to chemotherapy are challenging problems where angiogenesis
plays fundamental roles. Thus, targeting of VEGFR-2 signalling pathway has been an attractive approach. In
this study, we synthesised a new sorafenib analogue, thieno[2,3-d]pyrimidine based urea derivative, KM6. It
showed 65% inhibition of VEGF2 tyrosine kinase activity and demonstrated a potential antitumor activity in
TAM-resistant, LCC2, and its parental MCF7 BC cells. KM6 retained the sensitivity of LCC2 through upregula-
tion of key enzymes of apoptosis and proteins of cell death including caspases 3, 8, 9, P53, BAX/BCL-2 ratio
and LDH in media. It downregulated mRNA expression of Ki-67, survivin, Akt, and reduced levels of ROS and
glucose uptake. Moreover, KM6 reduced the levels of inflammation markers PGE2, COX2, IL-1b and IL6 and
metastasis markers MMP-2 and MMP-9. In conclusion, KM6 is a promising compound for ER þ and TAM-resist-
ant BC with many potential antitumor and polypharmacological mechanisms.
KEYWORDS
Thieno[2,3-d]pyrimidine-
based urea derivative;
breast cancer; VEGFR-2
kinase; apoptosis makers;
antiangiogenesis; anti-
inflammatory
GRAPHICAL ABSTRACT
Introduction
2012 to 22 million by 20222. BC involves a complex microenviron-
ment with unbalanced biological characteristics3. The majority of
The World Health Organisation considers breast cancer (BC) as the
most common malignant tumour among women1. It is expected
that the number of new cases will increase from 14 million in malignant breast tumours are classified as oestrogen receptor
CONTACT Samia A. Shouman
Fom El Khalig, Cairo, Egypt; Marwa Kamel
Fom El Khalig, Cairo, Egypt
samia.shouman@nci.cu.edu.eg
Department of Cancer Biology, National Cancer Institute, Cairo University, Kasr Al Eini Street,
marwawka@cu.edu.eg Department of Cancer Biology, National Cancer Institute, Cairo University, Kasr Al Eini Street,
Supplemental data for this article can be accessed here.
ß 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

1642
M SHARAKY ET AL.
positive (ERþ) BC where antiestrogens play a key role in the endo- that acts as a multityrosine kinase inhibitor. It inhibits VEGFR-2,
crine therapy of this type. The nonsteroidal compound Tamoxifen PDGFR, RAF, FLIT-3 and C-KIT and is used in treatment of hepato-
(TAM) is the most extensively used antiestrogen. However, cellular and renal cell carcinomas13. It has been investigated in
acquired resistance to TAM is a serious therapeutic problem the treatment of TAM-resistant BC and showed remarkable sensi-
among ER þ BC patients4. TAM resistance arises because of irregu- tisation to TAM14. By modification of sorafenib structure, several
lar signalling pathways within the tumour microenvironment and analogues have been synthesised to improve its cytotoxic and
it has been illustrated that angiogenesis plays a fundamental role antiangiogenic activities. These modifications include replacement
in this resistance5.
of urea with thiourea15, ether link with thioether16, pyridyl car-
boxamide moiety with other scaffolds as pyrazolo pyrimidine I
and quinazoline II17,18. Some of these analogues exhibited better
activity than sorafenib but none of them has been examined
against TAM resistance. Thieno [2,3-d]pyrimidine ring system con-
stitutes an attractive chemical scaffold with several reported bio-
logical activities e.g. inhibition of inflammatory-related diseases,
Angiogenesis ensures sufficient supply of oxygen and
nutrients to proliferating cells, resulting in tumour progression
and metastasis6. Tumour angiogenesis is regulated by a finely
tuned equilibrium between proangiogenic and antiangiogenic
factors, produced by host and tumour cells, including vascular
endothelial growth factor (VEGF)7. VEGF family of tyrosine kinase
receptors includes three protein receptors: VEGFR-1 (FLT1), acting as potent ligands for 5-HT3 and/or 5-HT4 receptors and
VEGFR-2 (KDR/FLK1) and VEGFR-3 (FLT4). VEGFR-1 and VEGFR-2 others19–22. Moreover, it is considered a bioisostere of 4-amino-
generally mediate angiogenesis while VEGFR-3 regulates lym- quinazoline core that comprises the structures of potent anti-
phangiogenesis. Specifically, suppression of VEGF/VEGFR signal- cancer drugs as gefitinib and erlotinib. Several literatures
ling pathway is a remarkable therapeutic target for inhibiting reported that thieno[2,3-d]pyrimidines fused with lipophilic cyclo-
tumour angiogenesis and subsequent tumour growth. VEGFR-2 is alkyl ring (III, IV) potentiate the anticancer activity23–25
.
the most important transducer of VEGF-dependent angiogenesis, Consequently, the present investigation is concerned with the
and therefore, treatment with VEGFR-2 inhibitors may be a way synthesis of a new sorafenib analogue KM6 with benzothienopyr-
to target tumour growth8,9 including BC10. In addition, it has midine ring system replacing the pyridyl carboxamide moiety in
been suggested that utilising VEGFR inhibitors is a way to over- sorafenib. The new derivative KM6 was tested for its cytotoxic
come endocrine resistance at multiple levels11. For example, ruxo- activity against different BC cell lines namely MCF7, T47D, MDA-
litinib has been proposed as a new therapeutic targeting agent MB and LCC2 (TAM-resistant cells). Additional experiments were
for TAM-resistant BC through inhibiting VEGF mRNA expression also performed to explore the different mechanisms where KM6
and transcriptional activity12. Sorafenib is a diaryl urea derivative exerts its cytotoxic activity.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1643
Drugs and compounds
Materials and methods
TAM citrate was obtained from (Amria Pharmaceuticals Company,
Alexandria, Egypt). It is a white powder stored at room tempera-
ture. TAM was dissolved in dimethylsulfoide (DMSO) and prepared
as stock solution at concentration 10 mM and was kept at ꢂ20 ꢁC.
Immediately before use, it was serially diluted in RPMI1640 to
yield a concentration range of 2–80 mM.
All starting materials, reagents and solvents were purchased from
(Sigma Aldrich Chemical Co., St. Louis) and used without further
purification. Monitoring of chemical reactions was performed by
analytical thin layer chromatography (TLC), on silica gel 60 F254
packed on Aluminium sheets, purchased from (Merck, New Jersey)
and were visualised using U.V. lamp (254 nm). Column chromatog-
raphy was conducted on silica 60 (35–70 microns) eluent gradient
hexane: ethyl acetate 90:10 to 85:15). Melting points were deter-
mined by (Stuart Scientific apparatus, UK). 1H-NMR spectra were
recorded on a Bruker 400 MHz spectrometer in d scale (ppm)
using DMSO as a solvent and TMS as an internal standard. 13C
NMR spectra was run at Joel 100 MHz spectrometer in d scale
J(Hz) using DMSO as a solvent and TMS as the internal standard.
The LC–MS-MS system consisted of a Agilent 1200 HPLC system
(Agilent technologies, CA) with a quaternary gradient pump
(Agilent 1260 infinity), an online vacuum degasser, a column oven
and an autosampler (Agilent 1260 infinity), coupled to a ABSCIEX
Q TRAP 3200 mass spectrometer (ABSCIEX, Germany) equipped
with an electrospray ionisation (ESI) interface. Data acquisition was
performed with analyst 4.0 software (ABSCIEX). Separation was
Synthesis and characterisation of KM6
As described in Schemes 1 and 2, synthesis of KM6 was accom-
plished by a convergent synthesis through condensation of the
diaryl urea derivative (1) with 4-chloro-5,6,7,8-tetrahydroben-
zo[4–5]thieno[2,3-d]pyrimidine derivative (4). The diaryl urea (1)
was obtained by reaction of p-aminophenol with 4-chloro-3(tri-
fluoromethyl) phenyl isocyanate in dry 1,4-dioxane28 (Scheme 1).
Derivative (4) was synthesised via three reaction steps starting by
reaction of cyclohexanone, ethyl cyanoacetate and elemental sul-
fur in the presence of piperidine (Gewald reaction) to give the 2-
amino tetrahydrobenzothiophene derivative (2). Compound (2)
upon cyclisation with formamide gave the tetrahydrobenzothieno-
pyrimidine derivative (3) which was chlorinated to afford com-
pound (4)29–31 (Scheme 2). 1H-NMR of KM6 revealed the
appearance of two exchangeable protons corresponding to NHs
at 9.8 and 10.0 ppm, aromatic protons at 7.08–8.34 ppm in add-
ition to cyclohexyl protons at 1.78, 2.9 and 3.0 ppm. 13C NMR
revealed the appearance of peaks at 168.03 (C4 pyrimidine),
163.51 (C2 pyrimidine), 153.10 (C ¼ O), 22.27, 22.84, 25.45, 25.94
(cyclohexyl protons). Quantification was performed with multiple
reaction monitoring (MRM) by using gas collision-induced dissoci-
ation and the following ion transitions: M/z 465.12/252.2 and
519.1/324.1 for sorafinib and KM6, respectively, with the cone vol-
tages all set at 45 V and the collision energy at 35 eV. Typical
performed using
a
Waters XBridge-C18-5 mm (2.1 ꢀ 150 mm
Column, Agilent, Germany) reversed phase analytical column
(Agilent, CA). The mobile phase was pumped at flow rate of
300 ml/min and it consisted of 0.1% formic acid/10 mM ammonium
format (solvent A) and 100% acetonitrile (solvent B). Over all run
time was 12 min. The mass spectrometer was operated in the
positive ESI mode with spray voltage set at 4 kV, at a temperature
of 350 ꢁC and a sweep gas flow of 20 L/h. Calculations were com-
pleted by the Multiquant software programme. Serial dilutions of
standards were prepared at concentrations ranging from 62.5 to
1000 ng/ml for sorafinib and KM6 to make calibration curves26,27
.
Scheme 1. Preparation of diaryl urea derivative: Reagents and conditions: (a) 1,4-dioxane, room temperature, 1 h, 75%.
Scheme 2. Preparation of KM6: Reagents and conditions: (a) S, piperidine, water bath at 50–60 ꢁC, 16h,70% (b) HCONH2, reflux, 3 h, 81% (c) POCl3, reflux, 3 h, 76%
(d) Acetonitrile, urea derivative (1), Cs2CO3, 60 ꢁC, 6 h, 66%.

1644
M SHARAKY ET AL.
chromatograms for detection of Sorafinib and KM6 are displayed using sigmoidal dose–response curve-fitting models (Graph Pad
in Supplementary Data SI. They were detected at retention times Prizm software, version 5).
0.98 and 11.1 min. Calibration curve was linear (r2 ꢃ 0.99).
In all the mechanistic experiments in this study, we used the
IC50 concentrations of TAM and KM6 in both cell lines. Therefore,
in MCF-7, 11 mM of TAM and 6.4 mM of KM6 were used, while in
LCC2 the concentrations used were 67.6 mM of TAM and 3.6 mM
of KM6.
A solution of the urea derivative (1) (0.147 g, 1 mmol) and cae-
sium carbonate (0.171 g, 2 mmol) in dry acetonitrile (10 ml) was
stirred for 1 h at room temperature. The 4-chloro thieno[2,3-d]pyr-
imidine derivative (4) (0.1 g, 1 mmol) was added slowly and the
reaction mixture was heated for 6 h at 55–60 ꢁC. The solvent was
removed under reduced pressure and the residue was stirred with
cold 4% NaOH solution (20 ml, 1 M) for 1 h. The solid product was
filtered and purified with column chromatography (eluent hexane:
ethyl acetate 90:10 to 85:15) to afford KM6 m.p. 250–253 ꢁC. MS:
(Mwt 518.94) (ES mass) (rel. int.) 519.08, (Mþ, 32%), 1H-NMR
(400 MHz, DMSO-d6) d: 10.08 (broad, 1H exchangeable NH), 9.83
(broad, 1H, exchangeable NH), 8.47 (s, 1H, pyrimidine), 8:16 (s, 1H,
Ar-H), 7.55–7.73 (m, 2H, ArH), 7.21 (d, 2H, J ¼ 8 Hz, Ar), 7.19 (d, 2H,
J ¼ 8 Hz, ArH), 3.02–3.33 (m, 2H, cyclohexyl), 2.8–2.9 (m, 2H cyclo-
hexyl), 1.87–1.91 (m, 4H, cyclohexyl). 13 C-NMR (100 MHz, DMSO-
d6) d: 168.0, 163.5, 153.1, 152.5, 147.1, 140.2, 137.5, 136.0, 132.4,
Molecular docking study
To get insights of the binding mode, interaction and orientation
of KM6 into the ATP binding site of VEGFR-2 kinase enzyme,
molecular docking simulation study was performed using C-
Docker protocol in Discovery Studio 2.5 Software to rationalise the
VEGFR-2 inhibitory activity of KM6.
To achieve this, the crystal structure of VEGFR-2 kinase enzyme,
complexed with sorafenib with PDB code (4ASD), revealed that
the substituted terminal phenyl ring lied in a deep extended
hydrophobic pocket formed by the movement of Phe1047 residue
of the “DFG” motif to induce the “DFG-out” conformation
(Figure 2(A)).
Validation of the C-Docker protocol used was performed by
redocking the lead compound (sorafenib) in the VEGFR-2 kinase
active site and aligning the lead compound’s bioactive X-ray con-
former with the best-fitted pose acquired from the docking run.
The alignment displayed good coexistence between both posed
with RMSD ¼ 0.95Aꢁ, demonstrating the capability of the used
docking protocol to fetch valid docking poses.
To execute the docking protocol, the protein structure was pre-
pared through protein preparation protocol that is integrated into
the software. Water molecules were preserved due to their role in
VEGFR-2/ligand interaction. The protein structure was also mini-
mised via SMART minimiser algorithm. Afterwards, binding pocket
and the surrounding amino acid residues were identified.
Compound (KM6) was prepared using ligand preparation protocol
of Accelry’s Discovery Studio. Finally, docking process was done
using C-Docker software in the interface of Accelry’s discovery stu-
dio 2.5. Ten docking poses of the docked compound (KM6) were
generated and examined carefully for selecting the best binding
mode similar to sorafenib in the VEGFR-2 binding pocket.
KM6 was shown to verify the essential key interactions, known
for type II VEGFR-2 inhibitors and comparable to sorafenib. Thus,
the thieno[2,3-d]pyrimidine moiety occupied the flat aromatic
hinge region and a hydrogen bond between N1- nitrogen and the
main chain NH group of Cys919 residue was observed. In addition,
the urea tail was engaged in three key hydrogen bonds with
Glu885 and Asp1046 residues. Finally, a Pi-cation interaction with
Lys868 residue, as well as a Pi-sigma interaction with Leu 840 resi-
due were established (Figure 2(B)). Its c-Docker interaction energy
was estimated to be equal to ꢂ48.5 kcal/mol which is comparable
to sorafenib ꢂ50.7 kcal/mol.
2
3
127.4, 127.0 (2 C), 126.0 (q, JCF ꢂ30.7 Hz), 123.3, 122.6 (q, JCF
¼
4.1 Hz), 120.0 (2 C),119.0 118.8, 117.1, 25.9, 25.4, 22.8, 22.2,
Elemental Analysis: Calcd for C24H18ClF3N4O2S; C 55.55; H 3.50; N
10.80 Found; C 55.41; H 3.53; N 10.63 (Supplementary data SI, SII,
SIII, SIV).
KM6:[1–(4-Chloro-3-trifluoromethylphenyl)-3–(4-((5,6,7,8-tetrahy-
drobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)oxy)phenyl)urea] was pre-
pared as stock solution at concentration 10 mM and was kept at
ꢂ20 ꢁC. Immediately before use, it was serially diluted in RPMI
1640 to yield a concentration range of 2–32 mM.
Cell lines
Human BC cell lines: MCF7, T47D and MDA-MB-231were obtained
from the American Type Culture Collection (ATCC, Minnesota,
USA) and were maintained at National Cancer Institute (NCI),
Cairo, Egypt in RPMI1640 medium containing 10% foetal bovine
serum and 1% penicillin-streptomycin. LCC2 was a generous gift
from Dr Robert Clarke (office of technology commercialisation,
Georgetown University, USA) and was cultured in EMEM medium
containing 5% charcoal foetal bovine serum and 1% penicillin–-
streptomycin. All cell lines were routinely incubated in 5% CO2 in
a humidified atmosphere at 37 ꢁC.
Biological evaluation
Cytotoxicity assay
The antitumor activities of KM6 and TAM on BC cells were eval-
uated by sulphorhodamine-B (SRB) assay32. Briefly, cells were
seeded at a density of 3 ꢀ 103 cells/well in 96-well microtiter
plates. They were left to attach for 24 h before incubation with
drugs. Next, cells were treated with different concentrations
2–32 mM of KM6, 2 ꢂ 14 mM of TAM for MCF7, T47D and MDA-MB-
231 cells and by 5–80 mM of TAM for LCC2. For each concentra-
tion, three wells were used and incubation was continued for
48 h. DMSO was used as control vehicle (1% v/v). At the end of
incubation, cells were fixed with 20% trichloroacetic acid, stained
with 0.4% SRB dye. The optical density (O.D.) of each well was
measured spectrophotometrically at 570 nm using ELISA micro-
plate reader (TECAN sunriseTM, Germany). The percentage of cell
survival was calculated as follows: Survival fraction ¼ O.D. (treated
Assessment of VEGF kinase activity
The in vitro enzyme inhibition determination for KM6 was carried
out in BPS Bioscience Corporation, San Diego, CA (www.bpsbio-
science.com). The VEGFR tyrosine kinase activity at single dose
concentration of 10 mM was performed, where VEGFR2 (KDR)
(BPS#40301) served as the enzyme source and Poly (Glu, Tyr)
sodium salt, (4:1, Glu:Tyr) (Sigma#P7244) served as the standar-
dised substrate. Kinase-Glo Plus Luminescence kinase assay kit
cells)/O.D. (control cells). The IC50 (concentration that produce (Promega, Madison, USA) was used according to the manufac-
50% of cell growth inhibition) value of each drug was calculated turer’s instructions.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1645
Assessment of VEGF level
Determination of nitric oxide (NO) content
MCF-7 and LCC2 cells were plated in 24 well plates with 2.5 ꢀ 104 Nitric oxide was determined in culture media of the control and
treated cells according to Miranda et al.34 Briefly, 0.5 ml cold abso-
cells/well. After treatment with drugs, the culture medium was
aspirated and centrifuged at 10,000 rpm at 4 ꢁC for 10 min and the
resultant supernatant was used for determination of VEGF by an
ELISA Human VEGF ELISA Kit (Koma Biotech Inc, Seoul, Korea,
Catalog No. K0331132) in accordance with the manufacturer’s
instructions. The amount of VEGF was expressed as pg/ml. Level
of VEGF was determined from calibration curve and the percent-
age of VEGF was calculated as follows:
lute ethanol was added to 250 ml culture media then left for 48 h
at 4 ꢁC to attain complete protein precipitation followed by centri-
fugation at 13,000 rpm for 1 h using cooling centrifuge. 100 ml of
nitrate standard solution were serially diluted in duplicate in a 96-
well microplate. Then, 100 ml of vanadium chloride were added to
each well rapidly followed by 50 ml sulphanilamide and 50 ml n-(1-
naphthyl) ethylene diamine in 2 N hydrochloric acid. The absorb-
ance at 540 nm was measured following incubation period of
30 min spectrophotometrically using an ELISA microplate reader
(TECAN SunriseTM, Germany). The level of total nitrite/nitrate was
expressed as mM supernatant media and determined using stand-
ard curve.
Level of VEGF ¼ ðLevel of treated cells=level of control cellsÞ
ꢄ 100
The level of VEGF was calculated using sigmoidal dose–res-
ponse curve-fitting models (GraphPad, Prizm5 software
incorporated).
Assay of caspases 3, 8 and 9 activity
Activities of caspases 3, 8 and 9 were measured spectrophotomet-
rically at 450 nm in cell lysate using Colorimetric Assay Kits
(Biovision, CA) (Cat numbers K106-25, K113-25 and K119-25,
respectively) following the manufacturer’s instructions. Percentage
change in caspase activity was determined by comparing the
results with the level of the untreated control. The experiment
was carried out three independent times.
Determination of malondialdehyde (MDA) content
Lipid peroxidation products were quantified by measuring MDA
level in cell culture lysate of control and treated cells using lipid
peroxidation (MDA) Assay Kit (Sigma Aldrich Chemical Co., St.
Louis, Catalog No. MAK085) following the manufacturer’s instruc-
tions. The absorbance was determined at 532 nm using a spectro-
photometer (Spectronic, Milton Roy Co.). The MDA concentration
was calculated using sigmoidal dose–response curve-fitting mod-
els (GraphPad, Prizm5 software incorporated).
Determination of glucose uptake in cell culture media
In the cell culture medium of the control and treated cells, the
concentration of glucose was determined using Colorimetric Assay
Kit (Randox, County Antrim, UK) (Cat No.GL 346) following the
manufacturer’s instructions. The absorbance was recorded at a
wavelength of 500 nm using a spectrophotometer (Spectronic,
The amount of MDA present in the samples was determined
from the standard curve.
C ¼ ðSa=SvÞ ꢄ D
Sa ¼ Amount of MDA in unknown sample (nmole) from stand-
A
A
sample
standard
Milton Roy Co.). Glucose conc. (mg/dl) ¼
ꢀ concentra-
ard curve
tion of standard
Sv ¼ Sample volume (ml) added into the wells
C ¼ Concentration of MDA in sample
D ¼ Sample dilution factor.
Determination of lactate dehydrogenase (LDH) in cell culture
media and cell lysate
Determination of superoxide dismutase (SOD)
In this study, LDH was measured in the media for the detection of
cytotoxicity (necrotic cell death). It was also measured intracellu-
larly after cell lysis as a marker for glycolytic pathway. Thus, MCF-
7 and LCC2 cells were plated in 24 well plates with 2.5 ꢀ 104 cells/
well. After treatment with drugs, the medium was aspirated, cen-
The activity of the antioxidative enzyme SOD was determined by
SOD determination kit (Sigma Aldrich Chemical Co., St. Louis, Cat
no 19160) in cell culture lysate of control and treated cells by fol-
lowing the manufacturer’s instructions. SOD level was calculated
relative to the corresponding protein content. The absorbance of trifuged at 10,000 rpm for 10 min at 4 ꢁC to remove any dead cells
the supernatant was determined at 450 nm using a spectropho- and was used for necrosis experiments. Cell pellets were lysed
using protein lysis buffer and used for the determination of LDH.
UV assay kit was used following manufacturer instructions
(Randox, County Antrim, UK, Cat no. LD 401). The activity was cal-
culated relative to the corresponding protein content. The absorb-
tometer (Spectronic, Milton Roy Co.). The SOD activity (inhibition
rate %) was determined using the equation:
È
ð
Þ
½ð Þ
Ablank 1 ꢂ Ablank 3
SOD activity inhibition rate %
¼
–
ð
Þ
ðAsample ꢂ Ablank 2Þꢅ= Ablank 1 ꢂ Ablank 3 g ꢀ 100
ance was recorded at 340 nm using
(Spectronic, Milton Roy Co.).
a spectrophotometer
LDH activity ðU=LÞ ¼ 4921 ꢀ ð 340 nm=minÞ
Determination of reduced glutathione (GSH) content
Reduced glutathione was determined adopting Ellman’s method33.
MCF7 and LCC2 cells were harvested, protein was precipitated
with trichloroacetic acid and Ellman’s reagent [5,5-dithiobis-(2-
nitrobenzoic acid)] (Sigma Aldrich Chemical Co, St. Louis) was
added to supernatant. GSH The absorbance was determined at
405 nm using spectrophotometer (Spectronic, Milton Roy Co.).
GSH content was calculated from standard calibration curve and
was expressed as mM using sigmoidal dose–response curve-fitting
models (GraphPad, Prizm5 software incorporated).
Determination of prostaglandin E2 (PGE2)
The concentration of PGE2 in cell culture media of the control
and treated cells was determined using the PGE2 Enzyme
Immunoassay Kit (R&D systems Inc, Minneapolis, (Cat No.
PKGE004B) following the manufacturer’s instructions. The concen-
tration of PGE2 was calculated in comparison with a plotted stand-
ard curve and was expressed as pg/ml.

1646
M SHARAKY ET AL.
Real-time polymerase chain reaction
containing 2.5% triton X-100 at room temperature, washed with
water and incubated overnight at 37 ꢁC in developing buffer
[5 mM CaCl2, 0.05% Brij 35, and 50 mMTris (pH 7.8)]. They were
stained with 0.5% coomassie brilliant blue R-250 for 1 h and then
destained in a 50% methanol and 10% acetic acid solution. Clear
bands represented areas of proteolytic activity. Human recombin-
ant MMP-2 and MMP-9 were loaded separately as positive
controls. Gels were scanned using image Scanner III LabScan 6.0.
To determine mean intensity of each band (mean pixel), the
band densities were measured with Win_Image Studio
Lite_5.2.5software. Data are represented as means SD.
Total RNA was extracted from cells using RNeasy Mini Kit (Qiagen,
Valencia, Netherlands). cDNA was prepared from RNA (200 ng) in a
20 ml reaction using High capacity cDNA archive kit (Applied
Biosystem, CA). Real-time PCR of GAPDH, COX-2, IL-1b, IL-6,
MMP2, MMP-9, Survivin, AKT and Ki-67 were performed in tripli-
cate on an ABI 7500 fast real-time PCR System using GoTaq PCR
master mix (Promega, Madison). Fast amplification parameters
were as follows: an initial denaturation step at 95 ꢁC for 10 min,
followed by 45 cycles of denaturing at 95 ꢁC for 15 s, annealing at
60 ꢁC for 1 min, then extension at 72 ꢁC. All primers used in this
study were purchased from Invitrogen (CA) (Supplementary Table
(SV)). Quantitative analysis of data was performed by using the
DDCt method35.
Statistical analysis
The cycle threshold (Ct) was determined automatically.
Data are represented as means SD. The data were analysed using
one-way analysis of variance (ANOVA) test. To assess the signifi-
cance of differences, the Tukey post hoc test was used. P values
less than 0.05 were statistically significant. Graphs were performed
using Prism software programme (graph pad prism software, ver-
sion 5, CA) and analysis of data was performed using GraphPad
InStat, version 5.
D CTð1Þðfor treated sample for gene of interest ðxÞÞ
¼ CT – CT
ðXÞ
ðGAPDHÞ
D CTð2Þðfor Control sample for gene of interest ðxÞÞ
¼ CT – CT
ðXÞ
ðGAPDHÞ
DD CT ¼ D CTð1Þ ꢂ D CTð2Þ
¼ relative expression ¼ DD CT ¼ D CTð1Þꢂ D CT
CT
2ꢂDD
ð2Þ
Results and discussion
KM6 shows cytotoxicity to different BC cell lines including
resistant cells
Agarose gel electrophoresis
Agarose gel electrophoresis was used for assessing BAX, BCL2 and
P53. Total RNA was extracted from cells using RNeasy Mini Kit
(Qiagen, Valencia, Netherlands). cDNA was prepared from RNA
(200 ng) in a 20 ml reaction using high capacity cDNA archive kit
(Applied Biosystem, CA). mRNA expression was measured by gel
electrophoresis and b-actin was used as internal control. Primer
sequences are displayed in Supplementary Table (SVI).
Both TAM and KM6 were tested for their cytotoxicity against TAM
sensitive MCF7 cells and TAM-resistant LCC2 cells. The IC50 of TAM
in LCC2 was around 6 folds higher than in MCF7 (67.6 0.07 mM
vs 11 0.05 mM) thus confirming the resistant phenotype of LCC2
cells (Figure 1(A,B)). Interestingly, KM6 displayed a more remark-
able antitumor activity than TAM on both MCF7 (IC50
¼
6.4 0.05 mM) and LCC2 (IC50 ¼ 3.6 0.03 mM) (Figure 1(C,D)).
This was in agreement with the study of Pedersen et al.14 who
reported preferential growth inhibition of TAM-resistant cell line
with the tyrosine kinase inhibitor, sorafenib due to reduction in
the total expression of oestrogen receptor a (ER a), phosphory-
lated ER, FOXA1 and AIB1 leading to re-sensitisation of TAM-resist-
ant cells to TAM. It is noteworthy that KM6 also demonstrated
remarkable cytotoxic activity against other BC cell lines T47D
(IC50 ¼ 6.9 0.04 mM) and MDA-MB-231 (IC50 ¼ 10 0.04 mM)
(Supplementary Figure (SVII)). In the subsequent experiments, we
aimed to study the different mechanisms whereby KM6 exerted
its cytotoxic activity in MCF-7 and LCC2 cell lines.
Determination of protein concentration
After incubation of control and treated cells for the specified times,
media were collected and stored at ꢂ80 ꢁC and cells were har-
vested by trypsinization then lysed with RIPA lysis buffer (25mM
Tris HCL pH 7.6, 150mM NaCl, 1% triton X-100, 1% sodium deoxy-
cholate and 0.1% SDS) containing protease inhibitors. Protein con-
centrations in media and cell lysate were determined by Bradford
Assay kit (Pierce, Rockford, IL). All experiments were run in triplicate.
Western blot analysis
After treatment of cells, cell pellet was resuspended in cold PBS and
subjected to centrifugation, the pellets were collected. The cell pel-
lets were lysed by resuspending in 1 ml complete RIPA buffer con-
taining protease/phosphatase inhibitor cocktail and placed on ice for
30 min till complete lysis. The lysate in RIPA buffer was transferred to
an Eppendorf and centrifuged for 15 min at 13,000 rpm at 4 ꢁC.
Extracted proteins were seperated by SDS-PAGE (12% acrylamide)
and blotted onto PVDF membranes. The membranes were probed
with IL-6 and b-Actin antibodies for normalisation. Band intensities
were quantified using Win Image Studio Lite_5.2.5 software using
b-actin as loading control. Data are represented as mean SD.
Effect of KM6 on angiogenesis
KM6 was evaluated for its ability to inhibit VEGFR-2 tyrosine kin-
ase enzyme activity in vitro at a single dose concentration of
10 mM as a measure to suppress angiogenesis. At such concentra-
tion, KM6 exhibited 65% VEGFR-2 kinase inhibition. Moreover,
KM6 decreased VEGF level in both MCF7 and LCC2 culture media
by 30.4 and 33%, respectively, compared to their corresponding
controls. This is similar to sorafenib and other analogues that is a
well-known inhibitor of VEGF in BC37–39. However, TAM alone
exhibited a more remarkable reduction of VEGF level in MCF7
than KM6 by 42.3% but no significant effect was observed on
VEGF in LCC2 cells (Figure 2(C)). This may be due to the antiestro-
genic nature of TAM in BC while oestrogens are known to
Gelatine zymography
MMP-2 and MMP-9 enzymatic activities in media of the control
and treated cells were determined by SDS-PAGE gelatine zymogra-
phy36. Gels were incubated for 15 min in renaturation buffer enhance VEGF expression40,41
.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1647
Figure 1. Cytotoxicity of TAM and KM6 on BC cell lines: (A, B) Effect of TAM on MCF7 and LCC2, respectively. (C, D) Effect of KM6 on MCF7 and LCC2, respectively, by
sulphorhodamine assay. Graphs and data analysis were performed using GraphPad InStat, version 5. The data represent means of at least three independent
a
experiments SD. Significantly different from control (P < 0.05).
KM6 downregulates Ki-67 mRNA in both MCF7 and LCC2 cells.
had anti-oxidant effect in LCC2 where it significantly increased
SOD by 33.6% and GSH by 56.7% while MDA and NO levels were
significantly decreased by 48.8% and 39.8%, respectively.
Compared to TAM, KM6 acts as a stronger prooxidant in MCF7
rather than the resistant cells. It’s noteworthy that previous
reports attributed a great deal of sorafenib cytotoxicity to the
The cell proliferation marker Ki-67 is a nuclear non-histone protein
present in all active phases of the cell cycle except in GO phase
and is a promising molecular target in the diagnosis of cancer42
.
Previous reports pointed to the role played by Ki-67 in the
molecular subtypes of BC43 as well as in TAM-resistant BC
patients44. Additionally, it was reported that sorafenib inhibits liver
regeneration in rats through a mechanism involving Ki-6745. Our
data indicated that TAM downregulated the expression of Ki-67 in
MCF7 while it induced a pronounced increase in its expression in
LCC2 cells. This effect may be due to the previously reported
molecular changes associated with the antagonistic activity of
TAM in MCF7, and the hyper-response of oestradiol in TAM-resist-
ant BC cell lines46. On the other hand, KM6 downregulated Ki-67
in both MCF7 and LCC2 (Figure 3).
generation of reactive oxygen species48 Thus, it can be speculated
.
that generation of free radicals contributes to the cytotoxicity of
KM6 in MCF7 rather than in LCC2 as in Figure 4(A–D).
KM6 affects apoptosis in both MCF7 and LCC2
KM6 upregulates caspases 3, 8 and 9 in MCF7 and LCC2
The effect of KM6 on the apoptotic caspases was investigated.
This included the caspases involved in the extrinsic pathway (cas-
pase 8), intrinsic pathway (caspase 9) and executioner pathway
(caspase 3). The results indicated that in MCF7, KM6 induced sig-
nificant increase in caspases 8, 9 and 3 by 53.8, 49 and 76.4%,
respectively. A similar pattern was observed in LCC2 where KM6-
treated cells displayed increase of all the studied caspases. It’s
observed that the induction of caspases by TAM is more pro-
nounced on MCF7 than KM6. However, LCC2 exhibited resistance
to the apoptotic effect of TAM. This resistance was not observed
in KM6-treated cells (Figure 5(A–C)). Previous studies reported
KM6 is pro-oxidant in MCF7 but anti-oxidant in LCC2
Cancer cells are characterised by an increase in the rate of reactive
oxygen species (ROS) production and an altered redox environ-
ment compared to normal cells. Most chemotherapeutics agents
raise intracellular levels of ROS, and can alter redox homeostasis
of cancer cells47. Consequently, KM6 was tested for its antioxidant
activity in both MCF7 and LCC2 cells. KM6 induced a pro-oxidant
state in MCF7 by significantly increasing the MDA and NO by
15.3% and 68%, respectively, accompanied by a significant reduc- that sorafenib exerts antiapoptotic effects through the caspase
tion of SOD by 28% and GSH by 53%. On the other hand, KM6 pathway in prostate cancer49.

1648
M SHARAKY ET AL.
Figure 2. (A) 2D interaction diagram of sorafenib co-crystallized with VEGFR-2 showing two hydrogen bonding with Cys919 (PDB ID: 4ASD) (B) Docking pose of KM6
into VEGFR-2 kinase enzyme. (C) Effect of TAM and KM6 on VEGF level in MCF7 and LCC2. Statistical significance of results was analysed using one-way ANOVA fol-
a
lowed by Tukey’s multiple comparison test. Significantly different from control, b from TAM and (p < 0.05). TAM was used at IC (11 mM for MCF7 and 67.6 mM for
50
LCC2). KM6 was used at IC (6.4 mM MCF7 and 3.6 mM for LCC2).
50
KM6 increases P53 mRNA and decreases survivin mRNA in MCF7
and LCC2
The effect of KM6 on survivin (member of the inhibitor of apoptosis
IAP family) was investigated as displayed in Figure 5(D). It was found
that survivin was decreased by 53.5 folds compared to control in
MCF7 and by 24 folds in LCC2 by KM6. TAM also reduced the
expression of survivin in MCF7 by 98-fold but increased its expres-
sion in LCC2. Concerning the tumour suppressor gene P53 which
plays an important role in apoptosis induction50, its gene expression
was significantly increased by KM6 by 19.5% in MCF7 and by 13.9%
in LCC2 compared to control (Figure 5(E)). This upregulation of P53
was previously observed by sorafenib in hepatocellular carcinoma51.
TAM, on the other hand, did not affect P53 expression in MCF7,
which is consistent with previous reports where TAM did not alter
P53 protein in MCF7 cells52. However, TAM reduced P53 expression
in LCC2 by 31% probably due to alteration of apoptotic responses
in resistant cells as previously described53.
Figure 3. Effect of TAM and KM6 on Ki-67 mRNA expression in MCF7 and
LCC2 by real time RT-PCR. Statistical significance of results was analysed using
one-way ANOVA followed by Tukey’s multiple comparison test. aSignificantly
different from control, b from TAM and (p < 0.05). TAM was used at IC50 (11
mM for MCF7 and 67.6 mM for LCC2). KM6 was used at IC50 (6.4 mM MCF7 and
KM6 increases BAX/BCL-2 ratio in MCF7 and LCC2
The current study indicated that KM6 favourably modulates the
3.6 mM for LCC2).
gene expressions of the apoptotic protein BAX as well as the

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1649
Figure 4. Effect of TAM and KM6 on oxidative stress markers (A) MDA, (B) NO, (C) SOD and (D) GSH in MCF7 and LCC2. Statistical significance of results was analysed
a
using one-way ANOVA followed by Tukey’s multiple comparison test. Significantly different from control, b from TAM and (p < 0.05). TAM was used at IC 50 (11 mM
for MCF7 and 67.6 mM for LCC2). KM6 was used at IC 50 (6.4 mM MCF7 and 3.6 mM for LCC2).
antiapoptotic proteins BCL-2. As shown in Figure 5(F–H), treat- KM6 modulates glucose metabolism through decreasing glucose
ment of MCF7cells by KM6 resulted in increasing BAX/BCL-2 ratio
by 4.56 folds in MCF7 and by 3 folds in LCC2. This was compar-
able to BAX/BCL-2 ratio produced by TAM in MCF7 (4.52).
However, in LCC2, KM6 was superior to TAM that decreased BAX/
BCL-2 ratio to 0.438%. Previous studies demonstrated that sorafe-
nib reduces the antiapoptotic protein Mcl-1, which is a member of
BCL-2 family leading to induction of apoptosis in lung cancer cell
line54 and it sensitises hepatocellular cancer cells to apoptotic
stimuli55. In addition, TAM downregulated BCL-2 in MCF7 but
without affecting BAX52, which is different from our study show-
ing slight increase in BAX.
uptake, LDH and AKT levels in both MCF7 and LCC2
Among the metabolic changes exhibited by tumour cells is an
increase in glucose metabolism and glucose dependence. Tumour
cells exhibit high levels of glycolysis even in the presence of suffi-
cient oxygen,
a phenomenon termed aerobic glycolysis or
Warburg effect. This involves increased glucose uptake, glycolytic
rates, ATP production, lactate as well as several intermediates and
enzymes that benefit rapidly proliferating cells. LDH is a key
enzyme in this process that converts most of glucose stores into
lactate. As a result, much of glucose metabolites are shifted from
simple energy production to the promotion of accelerated cell
growth and replication57. AKT is a key protein involved in PI3K-
AKT signal transduction pathway that promotes survival and
growth in response to extracellular signals. Dronamraju et al.58
reported that enhanced AKT signalling is associated with activa-
tion of various downstream enzymes involved in the glyco-
lytic process.
KM6 increases necrosis in both MCF7 and LCC2
A key sign for necrotic cells is the permeabilization of the plasma
membrane that can be detected in tissue culture by quantifying
the release of the intracellular enzyme LDH56. As depicted in
Figure 5(I), the LDH level in both MCF7 and LCC2 increased signifi-
cantly following treatment by KM6 by 37.2 and 78%, respectively.
TAM also induced necrosis in both cell lines, yet its effect on LCC2
was very pronounced where it increased by 177.1% compared to
control group.
In this study, treatment of MCF7 with KM6 resulted in decline
in both glucose uptake and LDH levels by 44 and 32.95%, respect-
ively, relative to their corresponding controls (Figure 6(A,B)). In
LCC2, a similar pattern was observed where glucose uptake was

1650
M SHARAKY ET AL.
Figure 5. Effect of TAM and KM6 on apoptotic and necrotic cell death. (A, B,C) Colorimetric assay of caspases Caspase-8, Caspase-9 and Caspase-3, respectively. (D)
mRNA expression of survivin determined by real-time RT-PCR (E) mRNA expression of P53 determined by agarose gel electrophoresis. (F, G) Agarose gel electrophoresis
of BAX and BCL-2, respectively. (H) BAX/BCL-2 ratio. (I) Effect of TAM and KM6 on necrosis as determined by measuring LDH levels in culture media of MCF7 and
LCC2. Band analysis was analysed by Win Image Studio Lite_5.2.5software. Statistical significance of results was analysed using one-way ANOVA followed by Tukey’s
multiple comparison test. aSignificantly different from control, bfrom TAM and (p < 0.05). TAM was used at IC 50 (11 mM for MCF7 and 67.6 mM for LCC2). KM6 was
used at IC 50 (6.4 mM MCF7 and 3.6 mM for LCC2).

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1651
Figure 5. (Continued).
reduced by 32.8% and LDH by 94.04%. This is in harmony with KM6 reduces the inflammatory mediators PGE2, COX2, IL-1b
previous data indicating that sorafenib curbs glucose utilisation in and IL6 in both MCF7 and LCC2
BC cells through the repression of mTORC1 activity59. It is notable
The inflammatory environment affects cancer cell behaviour,
including cancer formation, invasion and metastasis61.
Prostaglandin metabolism plays a pivotal role in inflammatory
processes and is proven to have a role in carcinogenesis, tumour
differentiation and tumour growth in BC. COX-2 is the key
involved enzyme while PGE2 acts as a ligand of the G-protein
coupled receptors EP2 which enhances VEGF expression associ-
ated with tumour neoangiogenesis62,63. Several cytokines also play
a role in tumorigenesis. For example, the inflammatory cytokine
IL-1b has been reported to induce the expression of markers asso-
ciated with malignancy in BC cells through Epithelial-
Mesenchymal Transition (EMT)64. In addition, the effect of IL-6
expression on EMT is mediated through activation of STAT365.
In this study, the potential effect of KM6 on several inflamma-
tory mediators (PGE2, COX-2, IL-1b and IL6) has been evaluated
(Figure 7(A–E)). The results demonstrated that a significant reduc-
tion of all the studied inflammatory markers was detected in both
cell lines. As compared to the control group, KM6 induced signifi-
cant decrease in PGE2 level by 33.7% in MCF7, and by 61.5% in
LCC2. A profound decline in COX-2 mRNA level was detected by
KM6 by 287.9% in MCF7 and by 843.1% in LCC2. KM6 also
that TAM was more effective than KM6 in reducing glucose
uptake in both MCF7 and LCC2 while the opposite was true in
case of LDH. Likewise, the results (Figure 6(C)) showed a signifi-
cant dramatic decrease in the gene expression of AKT by 232.4
folds in MCF7 and 106.9 folds for LCC2 as compared to control. In
addition, a profound decline in AKT expression was observed
compared to TAM-treated cells in both cell lines. It’s known that
activation of AKT is a mechanism of resistance to sorafenib in hep-
atocellular carcinoma60. In BC, the relation between AKT and sora-
fenib has not been widely investigated. However, it was
demonstrated that sorafenib inhibited cell proliferation and
induced apoptosis in a panel of BC cell lines with no correlation
to AKT59. Meanwhile, activation of PI3K/Akt signalling pathway
was found to mediate resistance to sorafenib in hepatocellular
carcinoma cells, and the combination of sorafenib and MK-2206,
an Akt inhibitor, overcame this resistance at clinical concentra-
tions60. Based on our results, KM6 displays a promising potential
in blocking AKT pathway and further studies are required to test
its ability to reverse acquired resistance to sorafenib especially in
hepatocellular carcinoma.

1652
M SHARAKY ET AL.
Figure 6. Effect of TAM and KM6 on glucose metabolism in MCF7 and LCC2. (A) Glucose uptake in media. (B): LDH level in cell lysate. (C) AKT mRNA expression.
Statistical significance of results was analysed using one-way ANOVA followed by Tukey’s multiple comparison test. aSignificantly different from control, bfrom TAM
and (p < 0.05). TAM was used at IC 50 (11 mM for MCF7 and 67.6 mM for LCC2). KM6 was used at IC 50 (6.4 mM MCF7 and 3.6 mM for LCC2).
induced a reduction in IL-1b and IL-6 by 9.9 and 4.7 folds in MCF7 invasion and metastasis of cancer cells through ECM degrad-
ation68. In BC tissues, MMP-2 and MMP-9 are highly expressed and
and by 155.7 and 33.4 folds in LCC2, respectively. Protein expres-
directly correlated to lymph node metastasis and tumour staging
and thus can be used as reference indices for guiding BC prog-
nosis and treatment69. Also, MMP-2 and MMP-9 have been impli-
cated in TAM resistance70. Our study shows the downregulation
of MMP-2 and MMP-9 gene expression and protein levels by
treatment with KM6 as indicated in Figure 8(A–D). Previous
reports about sorafenib similarly indicate that sorafenib sup-
pressed invasion in hepatocellular carcinoma cells through inhib-
ition of matrix metalloproteinase (MMPs) expression71. As far as
we know, the effect of sorafenib and its derivatives on MMPs in
BC has not been widely investigated. However, a previous study
concluded that the combination of sorafenib and radiation inhib-
its BC stem cells partly by reducing MMP-2 expression72. On the
other hand, as indicated by our data, TAM induced the upregula-
tion of both MMP-2 and MMP-9 in MCF7 and LCC2. This occurred
at the level of both gene expression as well as zymographic
activity measurements. This is a confirmation of previous data
where TAM-induced MMP-2/MMP-9 activities in ER þ PR þ human
BC cells73. However, as indicated by our data, their activities
were reduced in KM6-treated cells, which suggests that this drug
may have some antimetastatic activities. Given the multiple path-
ways of metastasis, further studies are needed to confirm
sion of IL-6 was also reduced in a similar pattern to mRNA expres-
sion following treatment by KM6.
The effect of sorafenib on inflammatory markers in BC has not
been widely explored. Recently, it was reported that sorafenib
improves therapy with alkylating agents by blocking inflammation,
invasion and angiogenesis in BC cells66. In addition, another study
verified that sorafenib inhibits signal transduction in response to
exogenous and endogenous cytokines67. Thus, in view of our
results, the sorafenib analogue KM6 exerts a promising anti-
inflammatory effect in BC.
An interesting observation is an obvious increase mediated by
TAM of all the studied inflammatory mediators in both MCF7 and
LCC2 and the ability of KM6 to reverse this inflammatory effect.
Further studies are thus required to elucidate the relation
between TAM and inflammation in BC as well as the potential use
of KM6 in modulating this effect.
KM6 downregulates migration and invasion markers MMP-2
and MMP-9 in both MCF7 and LCC2
The high expression of proteinases such as MMP-9 and MMP-2 in
the tumour microenvironment has a role in supporting migration, this mechanism.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1653
Figure 7. Effect of TAM and KM6 on inflammatory markers: (A) Effect on PGE2 using ELISA. (B–D) Effect on COX-2, IL-b and IL-6 mRNA expression as determined by
real time RT-PCR (E) Effect on protein expression of IL-6 as determined by western blotting. Statistical significance of results was analysed using one-way ANOVA fol-
a
lowed by Tukey’s multiple comparison test. Significantly different from control, bfrom TAM and (p < 0.05). TAM was used at IC 50 (11 mM for MCF7 and 67.6 mM for
LCC2). KM6 was used at IC 50 (6.4 mM MCF7 and 3.6 mM for LCC2).
Conclusion
through targeting several key enzymes and proteins involved in
From this study it can be concluded that the newly synthesised
thieno[2,3-d] pyrimidine based urea derivative KM6 is a potential
therapeutic agent for ER þ TAM sensitive and resistant BC, provid-
ing a basis for development of the compound as a novel anti-
tumour growth and progression. Through its polypharmacological
properties, KM6 can affect several pathways of angiogenesis,
redox activity, apoptosis, inflammation, glucose metabolism and
metastasis. Further preclinical, molecular and toxicologic studies
cancer agent. The mechanism of this activity is believed to be are needed to elucidate deeply the main mechanism of action

1654
M SHARAKY ET AL.
Figure 8. Effects of KM6 and TAM on invasion and metastatic markers in MCF7 and LCC2. MMP-2 and MMP-9 mRNA expression was determined by real time RT-PCR
in MCF7 (A), and LCC2 (B). Also, zymographic activity was determined in both cell lines (C, D). The analysis was done by WinImage Studio Lite_5.2.5software. Statistical
significance of results was analysed using one-way ANOVA followed by Tukey’s multiple comparison test. aSignificantly different from control, bfrom TAM and
(p < 0.05). TAM was used at IC 50 (11 mM for MCF7 and 67.6 mM for LCC2). KM6 was used at IC 50 (6.4 mM MCF7 and 3.6 mM for LCC2).
and to determine the optimal dose, route and formulation for
administering KM6.
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