Molecular docking, synthesis and anticancer activity of thiosemicarbazone derivatives against MCF-7 human breast cancer cell line

Article abstract of DOI:10.1016/j.lfs.2021.119305

Background: The aim of this study was to synthesize and evaluate anticancer activity of 2-hydroxy benzaldehyde and 4-hydroxy benzaldehyde thiosemicarbazone (2-HBTSc and 4-HBTSc) against MCF-7 breast cancer cell line. Materials and methods: The ligands were prepared and characterized by UV vis, IR and NMR. MTT assay was used to assess viability of cells. RNA isolation, extraction and cDNA synthesis were done. Then all groups were subjected to RT-qPCR using Gene expression specific primers. Also, western blot protein expression and molecular docking were done. Two-way ANOVA with Tukey post-hoc test was employed to test the significance using GraphPad Prism. Results: The IC₅₀ values were 3.36μg/ml and 3.60μg/ml for 2-HBTSc and 4-HBTSc treated MCF-7 tumor cells respectively. Tumor cell growth inhibition ranged from 38 to 49.27% in 4-HBTSc treated cells, and 19 to 25% in 2-HBTSc treated cells with increase in doses 5 μg/ml to 20 μg/ml. The protein and gene expression result showed a significant upregulation in tumor suppressor and apoptosis inducing genes while, oncogene activity was significantly downregulated. Specifically, BRCA2 and pRB gene showed the highest expression in 4-HBTSc and 2-HBTSc treated cells respectively. Conversely, RAS oncogene was downregulated significantly. Docking result showed that both 2-HBTSc and 4-HBTSc have the potential to inhibit Estrogen Receptor Alpha Ligand Binding Domain, Human 17-Beta-hydroxysteroid dehydrogenase type 1 mutant protein and Human Topoisomerase II alpha that are expressed more during Breast Cancer. Conclusion: The findings of this study imply that the test compound has potential for further study.

Full text of DOI:10.1016/j.lfs.2021.119305

Life Sciences 273 (2021) 119305
Contents lists available at ScienceDirect
Life Sciences
journal homepage: www.elsevier.com/locate/lifescie
Molecular docking, synthesis and anticancer activity of thiosemicarbazone
derivatives against MCF-7 human breast cancer cell line
a
b
a,
*
b
c
Belay Zeleke Sibuh , Sonia Khanna , Pankaj Taneja , Paratpar Sarkar , Neetu Kumra Taneja
a
Department of Biotechnology, School of Engineering and Technology, Sharda University, Greater Noida, U. P., India
b
Department of Chemistry and Biochemistry, School of Basic Sciences and Research, Sharda University, Greater Noida, U. P., India
c
Department of Basic and Applied Science, NIFTEM, Kundli, Sonipat, India
A R T I C L E I N F O
A B S T R A C T
Keywords:
Background: The aim of this study was to synthesize and evaluate anticancer activity of 2-hydroxy benzaldehyde
and 4-hydroxy benzaldehyde thiosemicarbazone (2-HBTSc and 4-HBTSc) against MCF-7 breast cancer cell line.
Materials and methods: The ligands were prepared and characterized by UV vis, IR and NMR. MTT assay was used
to assess viability of cells. RNA isolation, extraction and cDNA synthesis were done. Then all groups were
subjected to RT-qPCR using Gene expression specific primers. Also, western blot protein expression and mo-
lecular docking were done. Two-way ANOVA with Tukey post-hoc test was employed to test the significance
using GraphPad Prism.
Cytotoxicity
Thiosemicarbazone
MCF-7 breast cancer cell line
Molecular docking
2
4
-hydroxybenzaldehyde
-hydroxybenzaldehyde
Gene expression
Protein expression
Results: The IC50 values were 3.36
respectively. Tumor cell growth inhibition ranged from 38 to 49.27% in 4-HBTSc treated cells, and 19 to 25% in
-HBTSc treated cells with increase in doses 5 g/ml to 20 g/ml. The protein and gene expression result showed
μ
g/ml and 3.60 g/ml for 2-HBTSc and 4-HBTSc treated MCF-7 tumor cells
μ
2
μ
μ
a significant upregulation in tumor suppressor and apoptosis inducing genes while, oncogene activity was
significantly downregulated. Specifically, BRCA2 and pRB gene showed the highest expression in 4-HBTSc and 2-
HBTSc treated cells respectively. Conversely, RAS oncogene was downregulated significantly. Docking result
showed that both 2-HBTSc and 4-HBTSc have the potential to inhibit Estrogen Receptor Alpha Ligand Binding
Domain, Human 17-Beta-hydroxysteroid dehydrogenase type 1 mutant protein and Human Topoisomerase II
alpha that are expressed more during Breast Cancer.
Conclusion: The findings of this study imply that the test compound has potential for further study.
1
. Introduction
exerts its biological effect by binding to
α and β estrogen receptor (ER),
which are members of the superfamily nuclear receptor transcription
factors that are characterized by highly conserved DNA-and ligand-
binding domains. These nuclear receptors are important for the control
of gene expression. The mutation of this gene produces abnormal
version of the estrogen receptor or mutated protein also called ER+, the
expression of which results a breast cancer in female [6–11].
The biological activity of steroid hormones is regulated at the pre-
receptor level by several enzymes. Beta-hydroxy steroid dehydroge-
nase type 1 and type 2 plays an essential role in the activation of estrone
and inactivation of estradiol. 17 beta-hydroxysteroid dehydrogenases
(17β-HSDs) are important for the last step of estrogen and androgen
activation and the first step of their degradation. Any mutation in the
gene forming this product would switch the whole pathway and trigger
the formation of breast cancer. Breast cancer is due to over expression of
Cancer was the second leading cause of death globally in 2012 and
was responsible for 8.2 million deaths which increased to 9.6 million in
018. Globally, about 1 in 6 deaths is due to cancer and it is projected to
2
be 23.6 million new cases with 12 million deaths per year by 2030 [1–3].
Breast cancer is the most common type of cancer and the second leading
cause of death among women worldwide [4]. It affects 2.1 million
women per year, and an estimated 627,000 women died of breast cancer
in 2018, 15% of all deaths from female cancer. Although breast cancer
rates among women are higher in developed regions, rates increase
globally in nearly every country [5].
The primary female sex hormones, estrogens, can stimulate the
development, proliferation, migration, and survival of target cells, as
well as the development of secondary sexual characteristics. Estrogen
*
Corresponding author.
E-mail address: pankaj.taneja@sharda.ac.in (P. Taneja).
https://doi.org/10.1016/j.lfs.2021.119305
Received 22 December 2020; Received in revised form 24 February 2021; Accepted 25 February 2021
Available online 3 March 2021
0
024-3205/© 2021 Elsevier Inc. All rights reserved.

B.Z. Sibuh et al.
Life Sciences 273 (2021) 119305
Scheme 1. Preparation of 2-HBTSc and 4-HBTSc.
this protein in the cell surface of breast cancer tissue [12–16]. Also,
for the ligands, which were prepared by the reaction of MeOH and water
solutions with different substituent and thiosemicarbazide using the
procedures depicted below. The structure of the ligand was confirmed
from UV–Vis, IR and NMR spectral studies. UV Vis Spectroscopy was
performed by taking DMSO as reference sample or blank.
approximately 35% of Human epidermal growth factor receptor 2
(
HER2)-amplified breast cancers have co-amplification of the topo-
isomerase II-alpha gene encoding [17]. Topoisomerases are nuclear
enzymes that are essential to several processes such as transcription and
replication. They are important in regulating the structure of DNA by
facilitating the temporary cleavage and ligation cycle of DNA. Among all
forms of topoisomerases, topoisomerase II alpha domain has been
associated with cell proliferation and human endothelial receptor over
expression leading to breast cancer. Therefore it is an important thera-
peutic target of many anticancer therapy [18–21]. On the other hand,
p53 tumor suppressor gene is known as the guardian of the genome and
play an essential role in various signaling pathways to maintain the
integrity of genome [22]. It involves in cell cycle regulation, meta-
bolism, hormone-induced processes, transcription, DNA repair, epi-
genome, senescence and autophagy [22–26]. Consequently, most
tumorigenesis are associated with p53 gene mutation [27].
2.1.1. Synthesis of 2-hydroxybenzaldehyde thiosemicarbazone (2-HBTSc)
8 9 3
2-Hydroxybenzaldehyde thiosemicarbazone, C H N OS, was pre-
pared using previously reported general procedures [48] by refluxing
the mixture of 2-hydroxybenzaldehyde (2.33 ml, 0.02 mmol) and thio-
semicarbazide (1.82 g, 0.02 mmol) in a mixture of methanol (30 ml) and
distilled water(30 ml) as the solvent medium in the presence of glacial
acetic acid (2–3 ml) (Scheme 1a). The reaction mixture was refluxed for
about 3–4 h, resulting in a yellow-colored solution, which was kept
undisturbed. The next day, a crystalline yellow product separated out by
forming a layer which was filtered, washed with ethanol and dried.
◦
Yield, 4.046 g, melting point 259 C. Insoluble in: CHCl
3
, CCl
4
, CH
3
CN,
hiosemicarbazones are Schiff based ligands receiving significant
attention due to their versatile biological and pharmacological activ-
ities, metal DNA-interaction, structural diversity and availability of
bonding modes [28–30]. Thus, over the past years, thiosemicarbazone
and their derivatives have demonstrated a wide spectrum of biological
activity, viz. anti-fungal [31,32], anti-tumor [33–38], antimicrobial
2
H O; soluble in DMSO, DHF/THF.
2.1.2. Synthesis of 4-hydroxybenzaldehyde thiosemicarbazone (4-HBTSc)
4-Hydroxybenzaldehyde thiosemicarbazone (4-HBTSc) was pre-
pared using previously reported procedure [49] with slight modification
by refluxing a mixture of 4-hydroxybenzaldehyde (1.222 g, 0.01 mmol)
and thiosemicarbazide (0.914 g, 0.01 mmol) in a mixture of methanol
(15 ml) and distilled water (10 ml) as a solvent medium (Scheme 1b).
The reaction mixture was refluxed for about 5 h, resulting in a light-
yellow colored solution, which was kept undisturbed. Next day, a
light-yellow crystalline compound separated out by forming a layer and
[
39–41], and antiviral [42,43].
Studies have shown unwanted side effects of the existing cancer
chemotherapy and multidrug resistance contribute to a growing interest
in researching new anticancer agents, including synthetic compounds,
with reduced toxicity to normal tissue and less tumor cell resistance
[
44,45]. Since cancer is a complex and multiple gene-related disease, the
it was filtered, washed with ethanol and dried. Yield: 1.708 g, melting
◦
development of multi-targeted anticancer drugs to improve therapeutic
efficacy and reduce drug resistance is currently the focus of promising
research [33,46,47]. Therefore, this study is designed to synthesize,
perform molecular docking and evaluate in-vitro anticancer activity of
different thiosemicarbazones against MCF-7 human breast cancer cell
line.
point: 207 C; insoluble in CHCl
3
4 2
, CCl , H O, ethanol; soluble in DMSO,
DHF/THF, CH
3
CN and Methanol.
2.2. Biological studies
2.2.1. Cell lines and cell culture
The in vitro anticancer activity of the study compound was tested
against MCF-7 breast cancer cell line and for comparison, MCF-10
normal cell line was used. Initially, the cell lines were procured from
the National Centre for Cell Science, Pune (NCCS). Cells were cultured in
a 6 walled culture plate using Dulbecco’s Modified Eagle’s Medium, that
2
. Experimental section
2
.1. Chemistry
All chemicals used were commercial products of analytical reagent
was supplemented with 10% fetal bovine serum at 95% air condition
◦
grade procured from Sigma-Aldrich (Sigma Aldrich, MO, USA), except
with 5% of carbon dioxide (CO
2
) in 37 C Temperature.
2

B.Z. Sibuh et al.
Life Sciences 273 (2021) 119305
2
.2.2. MTT cell viability assay cell count
Cells were counted using hemocytometer (x10 cell/ml) before the
tumor cell lines were subjected to quantitative reverse transcription-PCR
(RT-qPCR) using Gene expression specific primers (Supplementary
Table 3) was done. GAPDH was used as a housekeeping gene and
selected 17 genes viz. 10 tumor suppressor genes, 4 oncogenes, 2-hor-
monal genes and 1 apoptosis inducing gene were tested for their
expression.
4
cell viability test conducted. Then cell viability was assessed by MTT
assay as described in [33,50,51] using ATCC MTT Cell Proliferation
Assay kit (ATCC, VA, USA). MCF-7 and MCF-10 cell lines were cultured
4
separately in 96 well plates at a concentration of 1 × 10 cells/well.
After 48 h the media was removed and cells were washed twice with
1
00
μ
l serum-free medium. Then 10
μ
l MTT Reagent was added and left
2.2.5. Western blot analysis
◦
for incubation for 4 h at 37 C in a CO2 incubator, until intracellular
purple formazan crystals are visible (until purple precipitate is visible).
After that, MTT containing medium was discarded, the cells were
Western blot is a commonly used analytical technique to detect
specific proteins in a given sample. Because an antibody is used to
specifically detect its antigen, the technique is also known as protein
immunoblotting. By analyzing the location and intensity of the band
formed, the expression details of the target proteins in the given cells
could be obtained [54]. Western blot analysis was performed using
previous established procedures [54,55], MCF-10A and MCF-7 cell lines
were cultured in DMEM/F12 Media with antibiotics till 70% confluency
stage. The groups were untreated, 2-HBTSc and 4-HBTSc at concentra-
washed with PBS (200
μl). The crystals were then dissolved by adding
1
00 l of DMSO and mixed properly by pipetting up and down and left at
μ
room temperature in the dark for 2 h. Spectro photometrical absorbance
of the purple blue formazan dye was measured in a micro-plate reader at
5
70 nm. Then, percent Cell viability was calculated using the formula
from Abdellatif et al. [46]
tion of 50 g/dish. The cells were harvested after 72 h of treatment.
μ
Mean absorbance of treated
Mean absorbance of untreated
%
Cell viability =
× 100.
Proteins were extracted with ice-cold EBC buffer with proteinase in-
hibitors. After gel-electrophoresis and transfer to nitrocellulose mem-
branes, proteins were visualized by immunoblotting with affinity-
purified polyclonal antibodies (Supplementary Table 4), followed by
incubation of the filters with HRP-conjugated secondary antibodies
purchased from Santa Cruz Biotechnology (Santa Cruz, Dallas, Texas,
USA), and reaction with the Enhanced chemiluminescent (ECL) detec-
tion kit from Sigma Aldrich (Sigma Aldrich, MO, USA).
2
.2.3. Evaluation of anticancer potential
The in vitro anticancer activities of the test ligands were studied for
short-period of time. First, 1 mg of the synthesized thiosemicarbazone
ligand was dissolved in 1 ml of Dimethyl sulfoxide (DMSO) separately.
The test was done by taking the desired doses directly (without further
dilution). Hence, the study was conducted in five concentrations
including the control group (i.e., 0, 5
μ
g/ml, 10
μ
g/ml, 12
μ
g/ml, 20 g/
μ
ml of the test ligand) for each of the cell lines used. Based on the pre-
pared test drug concentrations we have assigned the following groups.
2.3. Molecular docking study
Group I: the cell line was subjected only for 10
II: 5 g/ml of the test ligand; Group III, 10
Group IV, 12 g/ml test ligand; Group V, 20
μ
l DMSO (control); Group
g/ml of the test ligand;
Patch dock server-Bioinfo 3D, a molecular docking algorithm [56]
was used to carry molecular docking calculations of the both the syn-
thesized compound, 2-Hydroxybenzaldehyde thiosemicarbazone (2-
HBTSc) and 4-Hydroxybenzaldehyde thiosemicarbazone(4-HBTSc)
respectively with four protein receptors, Estrogen Receptor Alpha
ligand binding Domain,Y5375 Mutant (PDB id: 6CBZ), Human 17-Beta-
hydroxy steroid dehydrogenase type 1 mutant H221Q (PDB id: 1FDW),
Human Topoisomerase II alpha(5GWK) and Human P73 Tetrameriza-
tion domain(2WTT). All proteins were downloaded from Protein Data
Bank as PDB files (Supplementary Fig. 1–4). Water molecules attached
to these enzymes were removed so that they do not cause any interfer-
ence during the docking study. The structure of the synthesized 2-HBTSc
was drawn and cleaned using Chem Sketch software and a mol file was
generated which was then converted into PDB format. The PDB file of
synthesized 2-HBTSc was given a default PDB id of tclactvs000bBNjp.
pdb. And the other compound, 4-HBTSc was prepared using the previ-
ously stated method and a default PDB id tclactvs000ckTjaK.pdb was
given. Both these ligands were then docked into the active site of 4
different enzymes and protein 6CBZ, 1FDW, 5GWK and 2WTT one by
one. The docking was done at root mean square deviation (RMSD) of
atomic position value of 4 as the standard value. The docking results
using both the compounds for both targets were measured in the form of
binding energy.
μ
μ
μ
μg/ml test ligand.
2
.2.4. RNA isolation, cDNA synthesis and quantitative RT-PCR
The effects of 2-HBTSc and 4-HBTSc on the tumor cell were also
determined by molecular profiling. RNA was isolated and extracted
using previous procedure [52] through Trizol reagent (Thermo Fisher
Scientific, MA, USA) The sample was Lysed and homogenized in TRIzol
reagent. For homogenization initially, cells were centrifuged and the
growth media was discarded. Then, TRIzol reagent (0.75 ml of TRIzol
reagent per 0.25 ml of sample) was add directly to the culture dish to
lyse the cells and the lysate was mixed by vortex for homogenization.
After that, chloroform was added with ration of 0.2 ml of chloroform per
1
ml of TRIzol reagent used for lysis. Once the chloroform-containing
solution is added, the sample become cloudy. And it was vortexed for
1
5 s, incubated at room temperature for 5 min, and centrifuged (12,000
◦
×
g, 4 C, 15 min). Then the upper aqueous layer which contains RNA,
was transferred to a new tube. To precipitate RNA, 0.5 ml of isopropanol
to the aqueous phase, per 1 ml of TRIzol reagent used for lysis was
added. Then it was vortexed for 15 s, incubated at room temperature for
◦
1
0 min, and centrifuged (12,000 ×g, 4 C, 15 min.). Total RNA precip-
itate formed a white gel-like pellet at the bottom of the tube. To wash the
RNA, the supernatant was discarded and a volume of 75% ethanol equal
to the amount of TRIzol used during homogenization (step #1) was add.
2
.4. Statistical analysis
Then the sample was briefly vortexed and centrifuged for 5 min at 7500
◦
×
g at 4 C. After that discarding the supernatant and vacuum or air
Data is presented in the form of descriptive statistics through tables
drying of the RNA pellet for 10 min was done. Similarly, cDNA is syn-
thesized as described in [53] using Revert Aid First strand cDNA syn-
thesis kit (Thermo Fisher Scientific, MA, USA) through oligo-dT primers,
RiboLock RNase Inhibitor and Revert Aid reverse transcriptase in total
and graphs. Microsoft Excel was used for descriptive statistical analysis.
Whereas, IC50 value was calculated using Prism statistical software
package by fitting the sigmoidal dose response model. Also, a two-way
ANOVA and Mean comparison test using Tukey multiple comparison
test were analyzed in Prism statistical software package (GraphPad
reaction volumes of 20 L. Following that all groups of normal and
μ
3

B.Z. Sibuh et al.
Life Sciences 273 (2021) 119305
Fig. 1. IR spectra of 2-HBTSc and 4-HBTSc.
ꢀ
1
Software, Inc., La Jolla, CA, USA). P < 0.05* was considered statistically
υ
(C=S) and a broad band at 3421 cm to
υ
(O
–
H).
ꢀ 1
ꢀ 1
significant.
A sharp peak at 1540 cm and 1491 cm corresponded to
υ
(C=C)
ꢀ 1
and δ (NH
2
) respectively. A sharp peak at 1619 cm is associated to
ꢀ 1
ꢀ 1
3
. Results and discussion
υ(C=N). The medium peaks at 3317 cm and 3181 cm are assigned
to –
υ
(NH
2
) and (-N-H) respectively. In contrast, IR spectra of 4- HBTSc
υ
ꢀ
1
3
.1. Synthesis and characterization of thiosemicarbazones
(Fig. 1B) showed a sharp peak at 826 cm corresponding to
υ
(C=S) and
ꢀ 1
ꢀ 1
ꢀ 1
at 3470 cm due to
υ
(O
(C=C) and δ (NH
(C=N). The sharp peaks at 3239 cm and 3198 cm are assigned to –
(NH ) and (-N-H) respectively. The peaks are similar to reported
–
H). Peaks at 1586 cm and 1510 cm cor-
ꢀ 1
The synthesis of 2-hydroxy benzaldehyde thiosemicarbazone (2-
responded to
υ
2
) respectively and at 1619 cm to
ꢀ 1
ꢀ 1
HBTSc) and 4-hydroxy benzaldehyde thiosemicarbazone (4-HBTSc)
were carried out according to the steps depicted in experimental section
υ
υ
2
υ
(
Scheme 1). 2-HBTSc was a yellow crystalline solid, yield: 4.046 g,
values [57] and also confirmed the formation of 4-HBTSc.
1
◦
The
H NMR of 2-Hydroxy benzaldehyde thiosemicarbazone
melting point 259 C. Insoluble in: CHCl
3
, CCl
4
, CH
(N
) 1540(s), 1491(s);
3
CN, H
2
O; soluble in
ꢀ
1
DMSO, DHF/THF. IR (cm ):
H) 3186(m);
υ
(O
–
H)3421(b);
υ
–
H
2
) 3317(m);
υ
(-N-
(Fig. 2A) shows a singlet peaks at δ11.5 ppm corresponding to –OH and
at δ 9.85 ppm due to –NH proton. A multiplet of peaks is observed at δ
6.52–7.32 ppm because of the protons of the phenolic group. A singlet
peak is observed at δ 2.43 ppm due to –CH proton. These values are
υ
(C=N) 1619(s);
υ
(C=C) + δ (NH
2
υ
(
C=S) 833(s). 4-HBTSc was light yellow crystalline solid; yield: 1.708 g,
◦
melting point: 207 C; soluble in DMSO, DHF, CH
3
CN, Methanol and
ꢀ
insoluble in CHCl
(N ) 3239(s),
δ (NH ) 1586(s), 1510(s),1388(m);
-HBTSc (Fig. 1A) showed a sharp peak at 833 cm corresponding to
3
, CCl
(-N-H) 3198(s), 3150(m);
(C=S) 826(m)). The IR spectra of
4
, H
2
O, ethanol. IR (cm 1):
υ(O
–
H) 3470(s);
similar to the reported values. [57] (See Fig. 3.)
1
υ
–
H
2
υ
υ
(C=N) 1612(s);
υ
(C=C)
The
H NMR of 4-Hydroxy benzaldehyde thiosemicarbazone
+
2
υ
(Fig. 2B) shows a singlet peaks at δ11.22 ppm corresponding to –OH and
at δ 9.85 ppm due to –NH proton. A multiplet of peaks is observed at δ
ꢀ 1
2
4

B.Z. Sibuh et al.
Life Sciences 273 (2021) 119305
1
Fig. 2. H NMR spectra of 2-HBTSc and 4-HBTSc.
1
6
(
A) 2-HBTSc, H (DMSO- d , ppm):11.56 (s, H); 9.85 (s, H); 7.96, 8.01(d. 2H); 6.52–7.32 (m, 4H); 2.43 (s, 1H).
1 6
(
B) 4-HBTSc, H (DMSO- d , ppm):11.22 (s, H); 9.85 (s, H); 6.78, 6.72(d. 2H); 8.03–7.56 (m, 4H); 3.12 (s, 1H)
5

B.Z. Sibuh et al.
Life Sciences 273 (2021) 119305
Fig. 3. The effect of 2-HBTSc and 4-HBTSc on cell viability.
7
.56–8.02 ppm because of the protons of the phenolic group. A singlet
HBTSc. Interestingly, 4-HBTSc inhibited 38% growth of MCF-7 tumor
cells at the concentration of 5 g/ml, better than the highest inhibition
peak is observed at δ 3.12 ppm due to –CH proton. Similar result was
reported by. [49]
μ
(24.6%) by 2-HBTSc. Theoretically, the lowest IC50 is correlated with
the highest anticancer activity and the highest inhibition of cell growth
[
62]. Thus, the percentage of cell death due to cell growth inhibition is
3
3
.2. Biological evaluation
compatible with the IC50 value of the test ligands in the current study.
Similarly, MCF-10 normal cell line was used to determine whether the
test compounds were toxic or nontoxic to healthy cells. The IC50 values
of these ligands used in MCF-10 were higher than that of MCF-7. Overall,
these findings indicated that test compounds are selective in their
anticancer effect.
.2.1. In vitro anticancer activity of 2-hydroxybenzaldehyde
thiosemicarbazone and 4-hydroxybenzaldehyde thiosemicarbazone
The in vitro anticancer activity of 2-HBTSc and 4-HBTSc was tested
against MCF-7 breast cancer cell lines by using MCF-10A normal cells for
comparison. The half inhibitory concentration (IC50) of the test com-
pounds was calculated using GraphPad prism. In 2-HBTSc treated cells,
3.2.2. Effect of 4-HBTSc and 2-HBTSc on gene expression of MCF-7 breast
cancer cell line
the IC50 value is found to be 3.36
MCF-10A cell lines. Whereas, in 4-HBTSc treated cells, the IC50 is 3.60
g/ml and 9.55 g/ml for MCF-7 and MCF-10A cell lines respectively.
μ
g/ml for MCF-7 and 5.39
μ
g/ml for
The real time quantitative reverse transcription (RT-qPCR) and
ANOVA result (Tables 1 and 2) showed that the effect of 4-HBTSc and 2-
HBTSc on the mRNA expression levels of tumor suppressor genes were
significantly different from the mRNA expression levels in treated cells
as compared with MCF-7 control cells (p < 0.001). All tumor suppressor
genes were significantly upregulated except p19Arf which was down-
regulated. In 4-HBTSc treated cells, the highest level of expression
change was observed in BRCA2 followed by p53 and p19arf where they
were upregulated in 5.35, 4.715 and 3.125 folds respectively (Table 1
and Fig. 4A). Whereas, the lowest was observed in p21 where it was
upregulated by 1.59-folds. Similarly, pRB, BRCA1 and p53 have shown
the highest upregulation with 2.385, 1.78 and 1.005 folds respectively
in 2-HBTSc treated cells (Table and Fig. 4B). While p19Arf expression
was the lowest with a downregulation of 0.175 folds. In addition, this
study showed that treating MCF-7 breast cancer cell line with 4-HBTSc
and 2-HBTSc cause a significant downregulation of oncogenes than the
control and normal cells (Tables 1 and 2 and Fig. 4). RAS oncogene was
highly downregulated with 5.785 folds and 4.7 folds in 4-HBTSc and 2-
HBTSc treated cells respectively. On the contrary, upregulated expres-
sion of apoptosis inducing gene was observed in tumor cells treated with
both test compounds.
μ
μ
The MTT assay was used to test the cell viability. The result showed the
percentage of viable cells decreased with increasing in dose of treatment
(
Fig. 3a). The total viable cells found after the cells treated with different
doses of 2-HBTSc are 100, 81.1,76.81, 77.89, 75.36% in MCF-7 and 100,
7.61, 94.84, 94.04, 96.82% in MCF-10A cell lines when the cells are
treated with 0, 5 g/ml, 10 g/ml, 12 g/ml, 20 g/ml respectively
Supplementary Table 1). Similarly, 4-HBTSc has a highly significant (p
9
μ
μ
μ
μ
(
<
0.001) growth inhibition effect against MFC-7 tumor cell line
(
Fig. 5b). In MCF-7100, 62.31, 52.17, 51.81, 50.72% of cell were viable
and in MCF-10A, 100, 96.82, 93.25, 89.68, 84.92% of cells were viable
after they were treated with 0, 5 g/ml, 10 g/ml, 12 g/ml, 20 g/ml of
-HBTSc respectively (Supplementary Table 2).
-Hydroxy benzaldehyde and 4-hydroxy benzaldehyde are hydroxy
μ
μ
μ
μ
4
2
benzaldehyde isomers. In the first the hydroxy group located at the 2nd
carbon, while in the second, the hydroxy group is positioned at the 4th
carbon. It has been reported that structural variation, type of substitu-
ent, type of cancer and dose may affect the function of the compound
[
58–61]. The IC50 and the ANOVA findings in the current study also
confirmed that the ligand’s activity varied significantly due to substit-
uent position, dose and type of cell line. In MCF-7, the IC50 value of 2-
The result from RT-qPCR showed an improvement in the expression
of tumor suppressor genes which could be an indicator of the test
compound’s anticancer activity potential. Of the selected genes, the
treatment highly affected one of the major breast cancer susceptibility
gene BRCA2 (Table 1 and Fig. 4A). This gene involves in repair of DNA
HBTSc (3.36
μg/ml) is marginally lower than that of 4-HBTSc (3.60
μ
g/ml). However, when comparing the result of cell viability, 4-HBTSc
was more potent than 2-HBTSc. In line with this the highest percent-
age of growth inhibition 49.27%, 48.19%, 47.83% were observed when
the cells were treated with 20
μg/ml, 12
μ
g/ml and 10 g/ml of 4-
μ
6

B.Z. Sibuh et al.
Life Sciences 273 (2021) 119305
Table 1
Table 2
Effect of 2-HBTSc on the expression of selected genes.
Effect of 4-HBTSc on expression of different genes.
Name of
gene
Untreated (RQ
value)
Treated
(RQ
Fold change (MCF
7 treated ꢀ (MCF 7
untreated)
P value (P
< 0.05)
Name of
gene
Untreated (RQ
value)
Treated
(RQ
Fold change (MCF 7
treated) ꢀ (MCF 7
untreated)
P value (P
< 0.05)
value)
value)
MCF-
MCF-7
MCF-7
MCF-
10
MCF-
MCF-7
1
0
7
1
0
0
.06 ±
.06
2.16
6.88 ±
2.16
±
p53 (T.S)
4.715 upregulated
1.59 upregulated
1.06 ±
3.165 ±
± 0.02
0.045
p53 (T.S)
p21 (T.S)
1.005upregulated
0.49 upregulated
0
.06
0.045
.995
0.02
2.19
±
2
.19
0.02
3.78±
p21(T.S)
BRCA1
±
0.995
±
±
0.02
2.68 ±
0
.005
0
.03
0.99 ±
0.015
1.8 ±
4.21 ±
0.005
0.99 ±
0.015
0.02
1.8 ±
0.02
1.94
±
2
.405 upregulated
(
T.S)
BRCA2
T.S)
0.02
0.025
BRCA1
(T.S)
3.58 ±
1
.78 upregulated
1
.94
0.04
1.01 ±
7.29 ±
±
5.355 upregulated
3.125 upregulated
(
0.01
0.03
BRCA2
1.01 ±
2.2 ±
0
1
.035
.34
0.265 upregulated
(
T.S)
0.01
0.02
0.035
1.34
±
p19 Arf
T.S)
1.06±
4.46 ±
±
(
0.06
0.04
p19 Arf
T.S)
1.06±
1.16 ±
ꢀ 0.175
0
2
.015
.15
(
0.06
0.03
downregulated
0.015
2.15
±
PTEN (T.
S)
1.01 ±
4.37 ±
±
2.22 upregulated
1.885 upregulated
1.42 upregulated
0.01
0.035
PTEN (T.
S)
1.01 ±
3.095 ±
0
.025
0.95 upregulated
0.01
0.025
1
0
1
.05 ±
.045
.035
1.6 ±
0.02
0.73
±
3.48 ±
0.025
1.6 ±
0.02
0.73
±
pRB (T.S)
Bax (T.S)
0.025
pRB (T.
S)
1.05 ±
0.045
1.035
±
3.985 ±
2
.385 upregulated
0.025
2
0
.145 ±
±
.025
1.77 ±
0
.035
0.025
2.09
± 0.02
Bax (T.S)
1.04 upregulated
2.175 upregulated
0.78 upregulated
ꢀ 2.1 downregulated
0
.015
BCl2 (T.
S)
1.02 ±
0.02
5.145 ±
0.035
0.025
2.09
±
3
.055 upregulated
0.025
BCl2 (T.
S)
1.02 ±
4.26 ±
1
.055
Dmp1(T.
S)
2.49
6.085 ±
0.02
0.035
±
3.595 upregulated
0.02
2.49
±
± 0.02
0.035
0
1
.055
.015
1.055
±
Dmp1(T.
S)
3.27 ±
4
.84
0.04
2.095 ±
ꢀ 2.75
0.03
ER (Hor)
PR (Hor)
±
0.055
1.015
±
0.02
4.84
±
±
0.025
downregulated
0
0
.005
.995
2
.74 ±
ER (Hor)
PR (Hor)
1
0
.2 ±
1.5 ±
ꢀ 0.05
0.02
±
0.005
0.995
±
0.04
.015
0.035
downregulated
0
1
.005
.005
1
.2 ±
0.96 ±
ꢀ 0.24
9.83
±
4
0
.04 ±
ꢀ 5.79
0.015
0.01
downregulated
Ras (Onc)
Myc
±
0.005
1.005
±
.03
downregulated
0
0
.005
.955
0.035
9.83
±
Ras
5.13 ±
ꢀ 4.7 downregulated
7.45
2.78 ±
ꢀ 4.67
(Onc)
0.025
±
0.005
0.955
±
0.035
7.45
±
(
Onc)
± 0.03
0.02
downregulated
0
.045
Myc
3.23 ±
ꢀ 4.22
1
3.1
PKC
1.02 ±
4.21 ±
ꢀ 8.89
(Onc)
0.02
downregulated
±
0.045
0.03
13.1
±
(
Onc)
0.02
0.03
downregulated
0
.025
PKC
1.02 ±
8.23 ±
ꢀ 4.87
1
.065
Hdm2
7.24
4.295 ±
ꢀ 2.95
(Onc)
0.02
0.02
downregulated
±
0.025
7.24
±
(
Onc)
± 0.03
0.025
downregulated
0
.065
1.065
±
Hdm2
(Onc)
4.19 ±
ꢀ 3.05
Caspase-
3
1.005
±
0
0
.6 ±
2.365 ±
0.025
downregulated
1.765 upregulated
0.065
1.005
±
0.03
.02
0.035
(
apop)
0.005
Caspase-
3
0
0
.6 ±
1.93 ±
0
.0009***
1.33 upregulated
.02
0.03
(
Apop)
0.005
T.S = tumor suppressor; Hor = hormonal; Onc = oncogene; Apop = Apoptosis
0
.0005***
inducing.
T.S = tumor suppressor; Hor = hormonal; Onc = oncogene; Apop = Apoptosis
inducing.
double strand breaks and inter-strand crosslinks by RAD51-mediated
homologous recombination and mutation in this gene increases the
susceptibility to breast, ovarian and prostate cancers [63,64]. Likewise,
the other breast cancer susceptibility and tumor suppressor gene BRCA 1
was upregulated which could be due to functional similarity in their
encoded proteins [65] and work in a common pathway of genome
protection [63] with BRCA2. As mentioned above BRCA 2 involved in
DNA repair and cellular mechanisms, making it one of the targets of p53
gene, known as protector of the genome that plays a key role in many
signaling pathways to maintain genomic integrity [22]. Like BRCA2,
high expression of p53 gene was observed suggesting that the test drug
targets genes maintaining genomic integrity [66,67]. Recent review
[
68] indicated that co-inactivation of p53 and BRCA1 could lead to
tumor formation and development, and could be regarded as signatures
of molecular risk in cancer. Because, BRCA1 is coactivator of p53 [69]
and stabilize p53 using transcriptional activation of p14Arf by
enhancing p53-mediated transcription [70]. Conversely, p53 suppresses
the transcription of BRCA1, acting in a negative feedback loop of its own
stabilization [71]. Therefore, it is clear that the expression of p53 also
affects its regulator’s expression too. In line with this, higher mRNA
expression of p53 may be the explanation for increased expression of its
positive regulator PTEN and negative regulator p21, one of the
7

B.Z. Sibuh et al.
Life Sciences 273 (2021) 119305
Fig. 4. Effect of 4-HBTSc and 2-HBTSc on tumor suppressor, oncogene and apoptosis inducing gene expression.
transcriptional targets of p53, a cyclin-dependent kinases inhibitor, and
HDM2, a potent negative regulator of p53 [27]. The expression of HDM2
is up-regulated in many cancers, resulting in the loss of p53-dependent
activities such as apoptosis and cell-cycle arrest [72]. Reducing the ac-
tivity of HDM2 is therefore of critical importance for cancer therapy. The
compounds tested showed promising inhibitory activity on HDM2
indicating that there is potential for further study. Conversely, upregu-
lated expression of pRB was observed (Table 2 and Fig. 4B). Studies have
indicated that p53 and pRB play important role in maintaining genomic
stability and cell cycle regulation [73]. DNA damage activates p53
which promotes p21 transcription inhibiting CDKs that in turn phos-
phorylate pRB and block DNA synthesis [74]. Loss of pRB thus results in
deregulated cell proliferation and apoptosis, while loss of p53 de-
sensitizes cells to checkpoint signals, including apoptosis [27]. Thus,
improving the expression of pRB gene may lead to a healthy cell pro-
liferation and eventually enhance health and maintain genomic
stability.
survival tumor cells [76]. Finding a way to restore the function of these
RAS genes would therefore help to maintain the genomic stability and
reduce tumor development. In the present study RAS was down-
regulated by 5.78 and 4.7 (Tables1 and 2) folds when cells were treated
with 4-HBTSc and 2-HBTSc respectively. This finding indicated that
primary target for the test drugs could be the RAS gene. The interaction
between p53 and RAS signaling controls chemoresistance in cancer
[77]. In this study, an inverse relationship between p53 and RAS genes
was found, suggesting that the study compounds could be a multi-
targeting drug and could involve in signaling pathways that involve
both genes. However, further studies on different tumor cell lines with
multiple doses and signaling pathways should be conducted to reach
final conclusion. Similar finding was reported [77] indicating that p53
and RAS inversely regulate apoptosis through AKT- and ERK-mediated
signaling pathways. Another proto-oncogene Myc was downregulated
in treated cell lines. Myc is a master transcriptional regulator that con-
trols almost all cellular processes [78] and the deregulation of Myc leads
to development and progression of breast cancer and to other forms
cancer [79]. Thus, targeting Myc in therapy may help to avoid tumor cell
cycle. In our study, a downregulated and inverse relation was observed
between p53 and one of its transcriptional target c Myc. This inverse
RAS proteins are proto-oncogenes that are frequently mutated in
human cancers [75]. The Oncogenic mutations in RAS genes result in
increased cellular active RAS protein levels and increased signal prop-
agation through downstream pathways that drive proliferation and
8

B.Z. Sibuh et al.
Life Sciences 273 (2021) 119305
Fig. 5. Protein expression of selected genes and topoisomerase II.
relationship may be because the loss of p53 leads to Myc activation [80].
Similarly, estrogen receptor (ER) gene which are highly expressed in
breast cancer is downregulated by 2.75 and 2.1 folds in 4-HBTSc and 2-
HBTSc treated cells respectively. In addition, PKC oncogene was highly
downregulated by 8.89 folds in 4-HBTSc treated cells and 4.87 folds in 2-
HBTSc treated cells suggesting that PKC is also one of the main targets of
the studied compounds. Moreover, Caspase-3, a cysteine–aspartic acid
protease is responsible for cleaving cellular targets and induce cell death
[
81] was upregulated in 1.76 and 1.22 folds when the cell was treated
Fig. 6. (A) Docking interaction of amino acid residues of enzyme 1 FDW (Green
and Red color) with 4-HBTSc (Brown color). One acidic amino acid (Glutamic
caid) and the other amino acid (Valine) is involved in hydrogen bonding with
the ligand.
with 2-HBTSc and 4-HBTSc respectively. Overall, it can be concluded
that the test compounds have a potential for further study.
(
B) Interaction of amino acid residues of enzyme 1 FDW (Orange Color) with 2-
3
.2.3. Western blot analysis of protein expression
HBTSc (Yellow color). One acidic Serine residue (Orange) is involved in
hydrogen bonding (Black dotted line) with the ligand. (For interpretation of the
references to color in this figure legend, the reader is referred to the web
version of this article.)
As presented in Fig. 8, the protein expression confirmed what we
found in the gene expression. Tumor suppressor genes expression is
upregulated and the oncogenes expression are downregulated in treated
cell lines than the control. From tumor suppressor genes p53, pRB and
BRCA are highly upregulated. Also, Myc, HDM2, RAS and PKC oncogene
expression are highly inhibited indicating the drug could be used to
target oncogenes as well. Estrogen receptor (ER) expression is inhibited
in both treatment than the control. Besides, apoptosis inducing gene
3
.3. Molecular docking studies
The best binding mode of docked Ligand, 2-Hydroxy benzaldehyde
thiosemicarbazone (2-HBTSc) and 4-Hydroxy benzaldehyde thio-
semicarbazone (4-HBTSc) with Estrogen Receptor Alpha Ligand Binding
Domain (PDB Id: 6CBZ), Human 17-Beta-Hydroxy Steroid Dehydroge-
nase (PDB Id: 1FDW), Human Topoisomerase II alpha (5GWK) and
Human p73 Tetramerization domain (2WTT) is presented in Table 3 and
Fig. 6-9. The analysis of the molecular docking study helps to identify
different types of interactions such as H-bonding, hydrophobic and
electrostatic interactions. As presented in Tables 3, 4- HBTSc has
extended in to active site of 1FDW and interacted with 2 amino acids,
GLU 77 and VAL 79 (Fig. 6A). During this interaction, two hydrogen
(
Caspase-3) was upregulated. The expression of Topoisomerase II
α
(topo
II ), an important enzyme for DNA replication and is a target for anti-
α
cancer agents was studied. The result showed a decrement in the
expression of the enzyme which could be an indication that DNA
replication is inhibited in tumor cell lines after the treatment is applied.
Overall, all the protein expression is in line with the gene expression
and invitro anticancer activity studies. To better understand the
enzyme-inhibitor interactions and protein inhibitor interaction molec-
ular docking study was conducted.
9

B.Z. Sibuh et al.
Life Sciences 273 (2021) 119305
Fig. 7. (A) Docking Interaction of amino acid residues of cell surface receptor
6
CBZ (Red and white color) with 4-HBTSc (Yellow color). One acidic amino
acid (Glutamic acid) and the other amino acid (Valine) is involved in hydrogen
bonding with the ligand.
Fig. 8. (A) Docking Interaction of amino acid residues of 5GWK (Deep Blue,
Sky Blue and Ash Color) with 4-HBTSc (Dark green color). 3 hydrogen bonding
with basic amino acid (Arginine), 2 hydrogen bonding with acidic amino acid
(
B). Interaction of amino acid residues of 6CBZ (Green, Blue, Red and Light Blue
Color) with 2-HBTSc (Brown color). Basic amino acid (Asparagine), non-polar
amino acid Leucine and Iso Leucine and Lysine and acidic amino acid (Gluta-
mine) are involved in hydrogen bonding with the ligand. (For interpretation of
the references to color in this figure legend, the reader is referred to the web
version of this article.)
(
(
Glutamic acid) and one with non-polar amino acid (Alanine) were formed.
B) Docking Interaction of amino acid residues of 5GWK (Blue, Red and Ash
Color) with 2-HBTSc (Yellow color). Total of 5 hydrogen bonding with basic
amino acid (Arginine), 1 hydrogen bonding with acidic amino acid (Glutamic
acid) and one with non-polar amino acid (Alanine) were observed. (For inter-
pretation of the references to color in this figure legend, the reader is referred to
the web version of this article.)
bonds are formed and ꢀ 29.49 KCal/Mol binding energy is released.
Likewise, 2-HBTSc is extended into the active site of 1FDW (Fig. 6B) and
interacted with one amino acid SER 11 by forming one hydrogen bond
and binding energy of ꢀ 30.10 KCal/Mol is released. On the other hand,
when 4-HBTSc interacted with 6CBZ (Fig. 7A), there are two hydrogen
bond interaction with two amino acid, GLU 253 and GLY 420, and
Receptor Alpha Ligand Binding Domain Y5375 mutant (6 CBZ) with a
marginal binding free energy difference of ꢀ 0.06 kcal/Mol but this trend
was not observed when 4-HBTSc and 2- HBTSc was interacted with
1
FDW. In fact, 2-HBTSc is strongly interacted with Human 17-Beta-
-
23.78 KCal/mol binding energy is released. Whereas, in 6CBZ, 2-HBTSc
has interacted with total of 5 hydrogen bonds with five amino acids, GLU
23, LYS 449, ILE 386, LEU 387, ARG 394 and a binding energy of
26.91 KCal/Mol is released (Fig. 7B). The molecular docking analysis
indicates that 4-HBTSc binds stronger than 2-HBTSc with Estrogen
Hydroxysteroid dehydrogenase type I mutant (1FDW) than 4-HBTSc.
Hence, relatively more binding energy (ꢀ 0.61 kcal/mol more) is
released when 2-HBTSc binds with 1FDW. Similarly, the two other
proteins which are also taken for the study are Human Topoisomerase II
alpha (5GWK) and Human p73 Tetramerization domain(2WTT). When
3
ꢀ
1
0

B.Z. Sibuh et al.
Life Sciences 273 (2021) 119305
Fig. 9. (A) Docking Interaction of amino acid residues of 2WTT (Green color) with 4-HBTSc (Brown color). Two hydrogen bonding were formed with non-polar
amino acid (Leucine),
(
B) Interaction of amino acid residues of 2WTT with 2-HBTSc. No hydrogen bonding was found. (For interpretation of the references to color in this figure legend, the
reader is referred to the web version of this article.)
4
-HBTSc is docked with 5 GWK (Fig. 8A), a binding energy of ꢀ 29.67
activity was inhibited. The study suggested 4-hydroxy benzaldehyde
thiosemicarbazone can be used to target tumor suppressor genes and
oncogenes simultaneously, whereas, 2-hydroxy benzaldehyde thio-
semicarbazone is suitable for oncogene targeting experiments. In addi-
tion, the docking analysis confirmed that both 2-hydroxy benzaldehyde
and 4-hydroxy benzaldehyde thiosemicarbazone have the potential to
inhibit 1 FDW and 6 CBZ respectively. While 4-HBTSc can be used as a
potential candidate for Human Topoisomerase II alpha (5GWK) inhibi-
tion than 2-HBTSc. Overall, it can be concluded that 4-HBTSc have the
more potential to be better drug candidate for further preclinical studies
in-vitro and/or in-vivo using animal models.
Kcal/mol is released as compared with binding energy of ꢀ 28. 66 Kcal/
mol with 2-HBTSc (Fig. 8B). Similarly, a binding energy of ꢀ 25.66 Kcal/
mol is released when 4-HBTSc undergoes docking with 2WTT (Fig. 9A)
whereas, when its counterpart 2-HBTSc is docked with 2WTT, no
hydrogen bonding interaction has been established (Fig. 9B). Overall, it
can be concluded that both the synthesized compound (2-HBTSc and 4-
HBTSc) have proven to inhibit both 1FDW and 6CBZ which are highly
expressed in breast cancer respectively. While 4-HBTSc may act as a
potential candidate for Human Topoisomerase II alpha (5GWK) inhibi-
tion compared with 2-HBTSc.
Declaration of competing interest
4
. Conclusion
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
This study confirmed that, the activities of the ligands were signifi-
cantly varied due to type of substituent, dose and cell line. Tumor sup-
pressor genes were upregulated, while oncogenes were downregulated.
Similarly, the expression of apoptosis inducing gene was upregulated. In
agreement with the gene expression study, protein expression confirmed
that tumor suppressor genes were highly affected. Also, topoisomerase II
1
1

B.Z. Sibuh et al.
Life Sciences 273 (2021) 119305
Table 3
Docking result of 2-HBTSc and 4- HBTSC with 1FDW, 6CBZ, 5GWK and 2WTT.
Ligand
L)
Enzyme (E)
(PDB id)
Amino acid involved in
interaction (A.A)
Type of hydrogen bond interaction between ligand
Binding free energy (kcal/mol) of the best
Bond length
(A)
(
and enzyme (L … A.A of E)
docking interaction
2
4
-HBTSc
-HBTSc
1FDW
1FDW
SER 11
O—H
ꢀ 30.10
3.0954
2.63873
2.807
GLU 77
VAL 79
GLU 253
GLY 420
O—H-N
O—H-N
O—H-N
O—H-N
ꢀ 29.49
3.50893
3.41747
4
-HBTSc
6CBZ
ꢀ 26.97
2
2
2
3
2
2
.70576
.90399
.6581,
GLU 323
LYS 449
ILE 386
LEU 386
ARG 394
O———H-N
N-H——O
2
-HBTSc
6CBZ
O——H-O, O—H-N
N-H——O
ꢀ 26.91
.0145
.7379
N-H——N, N—H-N
.7627 3.375
ARG 804
ARG 804
ARG 804
GLN 746
GLN 746
ALA 745
GLU 746
ARG 804
N—H-N
N—H-N
N—H-N
O——H-N
O—H-N
O-H—O
O——H-N
N——H-N
N—H-N
N—H-N
N—H-N
O——H-N
N-H—O
O-H——N
–
3
2
.127619
.56911
4
2
-HBTSc
-HBTSc
5GWK
5GWK
ꢀ 29.67
ꢀ 28.66
3.11557
3
3
.58077
.16757
3.34779
3.549
3.09898
2.58708
3.41434
3.4716
3.56085
2.4346
–
ALA 745
4
2
-HBTSc
-HBTSc
2WTT
2WTT
LEU 385
ꢀ 25.27
No interaction
–
Acknowledgement
06 biological sciences 0604 genetics, Exp. Hematol. Oncol. 7 (2018) 1–11, https://
doi.org/10.1186/s40164-018-0116-7.
[
9] S.C. Hewitt, K.S. Korach, Estrogen receptors: new directions in the new
millennium, Endocr. Rev. 39 (2018) 664–675, https://doi.org/10.1210/er.2018-
00087.
The authors would like to acknowledge Sharda university for their
material support. Also, we thank Prof. Rajiv Gupta and Ms. Sanya
Pasichia, research fellow, Delhi University for their help during NMR
study.
[
[
10] L. Wang, G. Nanayakkara, Q. Yang, H. Tan, C. Drummer, Y. Sun, Y. Shao, H. Fu,
R. Cueto, H. Shan, T. Bottiglieri, Y.F. Li, C. Johnson, W.Y. Yang, F. Yang, Y. Xu,
H. Xi, W. Liu, J. Yu, E.T. Choi, X. Cheng, H. Wang, X. Yang, A comprehensive data
mining study shows that most nuclear receptors act as newly proposed
homeostasis-associated molecular pattern receptors, J. Hematol. Oncol. 10 (2017),
1
68, https://doi.org/10.1186/s13045-017-0526-8.
Funding source
11] J. Saczko, O. Michel, A. Chwi, E. Sawicka, M. Justyna, J. Kulbacka, R.W. Schulz, H.
F. Vischer, J.E.B. Cavaco, E.M. Santos, C.R. Tyler, H.J.T. Goos, J. Bogerd,
M. Brufani, F. Ceccacci, L. Filocamo, B. Garofalo, R. Joudioux, A. La Bella,
F. Leonelli, L.M. Migneco, R.M. Bettolo, P.M. Farina, G.S. Ashcroft, C. Routley,
M. Hardman, C. Meda, G. Rando, A. Maggi, Transport across natural and modified
biological membranes and its implications in physiology and therapy, Mol. Pharm.
This research did not receive any specific grant from funding
agencies in the public, commercial, or not-for-profit sectors.
2
27 (2017) 543–556, https://doi.org/10.1007/978-3-319-56895-9.
Appendix A. Supplementary data
[
[
12] X.Q. Wang, J.A. Aka, T. Li, D. Xu, C.J. Doillon, S.X. Lin, Inhibition of 17beta-
hydroxysteroid dehydrogenase type 7 modulates breast cancer protein profile and
enhances apoptosis by down-regulating GRP78, J. Steroid Biochem. Mol. Biol. 172
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.lfs.2021.119305.
(
2017) 188–197, https://doi.org/10.1016/j.jsbmb.2017.06.009.
13] C.Y. Zhang, W.Q. Wang, J. Chen, S.X. Lin, Reductive 17beta-hydroxysteroid
dehydrogenases which synthesize estradiol and inactivate dihydrotestosterone
constitute major and concerted players in ER+ breast cancer cells, J. Steroid
Biochem. Mol. Biol. 150 (2015) 24–34, https://doi.org/10.1016/j.
jsbmb.2014.09.017.
References
[
[
1] W.H.O. World Health Organization, Cancer. https://www.who.int/news-room/fa
ct-sheets/detail/cancer, 2020. (Accessed 29 August 2020).
2] J. Ferlay, M. Colombet, I. Soerjomataram, C. Mathers, D.M. Parkin, M. Pi n˜ eros,
A. Znaor, F. Bray, Estimating the global cancer incidence and mortality in 2018:
GLOBOCAN sources and methods, Int. J. Cancer 144 (2019) 1941–1953, https://
doi.org/10.1002/ijc.31937.
[
14] C.Y. Zhang, J. Chen, D.C. Yin, S.X. Lin, The contribution of 17beta-hydroxysteroid
dehydrogenase type 1 to the estradiol-estrone ratio in estrogen-sensitive breast
cancer cells, PLoS One 7 (2012) 1–7, https://doi.org/10.1371/journal.
pone.0029835.
15] J.A. Aka, M. Zerradi, F. Houle, J. Huot, S.X. Lin, 17Beta-hydroxysteroid
dehydrogenase type 1 modulates breast cancer protein profile and impacts cell
migration, Breast Cancer Res. 14 (2012), R92, https://doi.org/10.1186/bcr3207.
16] R. Mindnich, G. M o¨ ller, J. Adamski, The role of 17 beta-hydroxysteroid
dehydrogenases, Mol. Cell. Endocrinol. 218 (2004) 7–20, https://doi.org/10.1016/
j.mce.2003.12.006.
17] M.F. Press, G. Sauter, M. Buyse, L. Bernstein, R. Guzman, A. Santiago, I.
E. Villalobos, W. Eiermann, T. Pienkowski, M. Martin, N. Robert, J. Crown, V. Bee,
H. Taupin, K.J. Flom, I. Tabah-Fisch, G. Pauletti, M.A. Lindsay, A. Riva, D.
J. Slamon, Alteration of topoisomerase II-alpha gene in human breast cancer:
association with responsiveness to anthracycline-based chemotherapy, J. Clin.
Oncol. 29 (2011) 859–867, https://doi.org/10.1200/JCO.2009.27.5644.
18] N.D. Atkin, H.M. Raimer, Y.H. Wang, Broken by the cut: a journey into the role of
topoisomerase ii in DNA fragility, Genes (Basel) 10 (2019), https://doi.org/
[
[
[
[
3] J. Ferlay, I. Soerjomataram, R. Dikshit, S. Eser, C. Mathers, M. Rebelo, D.M. Parkin,
D. Forman, F. Bray, Cancer incidence and mortality worldwide: sources, methods
and major patterns in GLOBOCAN 2012, Int. J. Cancer 136 (2015) E359–E386,
https://doi.org/10.1002/ijc.29210.
[
[
[
[
[
4] E. Iorns, K. Drews-Elger, T.M. Ward, S. Dean, J. Clarke, D. Berry, D. El Ashry,
M. Lippman, A new mouse model for the study of human breast cancer metastasis,
PLoS One 7 (2012), https://doi.org/10.1371/journal.pone.0047995.
5] W.H.O. World Health Organization, Breast cancer. https://www.who.int/cancer
/
2
prevention/diagnosis-screening/breast-cancer/en/, 2018. (Accessed 23 January
020).
6] N. Fuentes, P. Silveyra, Estrogen receptor signaling mechanisms, in: Adv Protein
Chem Struct Biol, 2019, pp. 135–170, https://doi.org/10.1016/bs.
apcsb.2019.01.001.
7] Z.-R. Tang, R. Zhang, Z.-X. Lian, S.-L. Deng, K. Yu, Estrogen-receptor expression
and function in female reproductive disease, Cells 8 (2019) 1123, https://doi.org/
[
[
1
0.3390/genes10100791.
19] T. Liu, H. Zhang, S. Yi, L. Gu, M. Zhou, Mutual regulation of MDM4 and TOP2A in
cancer cell proliferation, Mol. Oncol. 13 (2019) 1047–1058, https://doi.org/
1
0.3390/cells8101123.
1
0.1002/1878-0261.12457.
8] H. Hua, H. Zhang, Q. Kong, Y. Jiang, Mechanisms for estrogen receptor expression
in human cancer 11 medical and health sciences 1112 oncology and carcinogenesis
1
2

B.Z. Sibuh et al.
Life Sciences 273 (2021) 119305
[
20] T. Chen, Y. Sun, P. Ji, S. Kopetz, W. Zhang, Topoisomerase II
α
in chromosome
and their nickel(II) and copper(II) complexes, J. Inorg. Biochem. 90 (2002)
113–126, https://doi.org/10.1016/S0162-0134(02)00428-2.
instability and personalized cancer therapy, Oncogene 34 (2015) 4019–4031,
https://doi.org/10.1038/onc.2014.332.
21] Y. Ali, S. Abd Hamid, Human topoisomerase II alpha as a prognostic biomarker in
cancer chemotherapy, Tumor Biol. 37 (2016) 47–55, https://doi.org/10.1007/
s13277-015-4270-9.
22] T.V. Denisenko, A.D. Pivnyuk, B. Zhivotovsky, P53-autophagy-metastasis link,
Cancers (Basel) 10 (2018) 1–20, https://doi.org/10.3390/cancers10050148.
23] I. Uehara, N. Tanaka, Role of p53 in the regulation of the inflammatory tumor
microenvironment and tumor suppression, Cancers (Basel) 10 (2018), https://doi.
org/10.3390/cancers10070219.
24] D.E. Moulder, D. Hatoum, E. Tay, Y. Lin, E.M. McGowan, The roles of p53 in
mitochondrial dynamics and cancer metabolism: the pendulum between survival
and death in breast cancer? Cancers (Basel) 10 (2018) https://doi.org/10.3390/
cancers10060189.
25] E.M. McGowan, Y. Lin, D. Hatoum, Good guy or bad guy? The duality of wild-type
p53 in hormone-dependent breast cancer origin, treatment, and recurrence,
Cancers (Basel) 10 (2018) 7–9, https://doi.org/10.3390/cancers10060172.
26] T. Oikawa, Y. Otsuka, H. Sabe, P53-dependent and -independent epithelial
integrity: beyond miRNAs and metabolic fluctuations, Cancers (Basel) 10 (2018),
https://doi.org/10.3390/cancers10060162.
[43] J.S. Chandra, Y.A.S.J.P. Kumari, P.N.V.V.L.P. Rani, Y. Sunandamma,
Anthraquinone benzylthiosemicarbazone Cr(III) complex as a potential anti-cancer
drug - characterization and activity, Indian J. Adv. Chem. Sci 2 (2013) 32–37.
[44] M. Hajrezaie, M. Paydar, C.Y. Looi, S.Z. Maghadamtousi, P. Hassandarvish, M.
S. Salga, H. Karimian, K. Shams, M. Zahedifard, N.A. Majid, H.M. Ali, M.
A. Abdulla, Apoptotic effect of novel Schiff based, Sci. Rep. 5 (2015) 1–11, https://
doi.org/10.1038/srep09097.
[
[
[
[45] S. Marsh, H.L. McLeod, Pharmacogenetics and oncology treatment for breast
cancer, Expert. Opin. Pharmacother. 8 (2007) 119–127, https://doi.org/10.1517/
14656566.8.2.119.
[46] K.R.A. Abdellatif, A. Belal, M.T. El-Saadi, N.H. Amin, E.G. Said, L.R. Hemeda,
Design, synthesis, molecular docking and antiproliferative activity of some novel
benzothiazole derivatives targeting EGFR/HER2 and TS, Bioorg. Chem. 101
(2020), 103976, https://doi.org/10.1016/j.bioorg.2020.103976.
[47] W. Zheng, Y. Zhao, Q. Luo, Y. Zhang, K. Wu, F. Wang, Multi-targeted anticancer
agents, Curr. Top. Med. Chem. 17 (2017) 3084–3098, https://doi.org/10.2174/
1568026617666170707124126.
[48] J.S. and E.R. Archana, Synthesis characterization and application of
salicylaldehyde thiosemicarbazone and its metal complexes, Int. J. Res. Chem.
Environ. 2 (2012) 130–148. http://www.ijrce.org/issue/october 2012/130-148.
pdf.
[49] S.K. Padmanabha, Spectrophotometric Method for the Determination of Trace
Amount of Bismuth in Alloy Samples Using vol. 4, 2011, pp. 857–862.
[50] K. Dhahagani, S. Mathan Kumar, G. Chakkaravarthi, K. Anitha, J. Rajesh, A. Ramu,
G. Rajagopal, Synthesis and spectral characterization of Schiff base complexes of
Cu(II), Co(II), Zn(II) and VO(IV) containing 4-(4-aminophenyl)morpholine
derivatives: antimicrobial evaluation and anticancer studies, Spectrochim. Acta -
Part A Mol. Biomol. Spectrosc. 117 (2014) 87–94, https://doi.org/10.1016/j.
saa.2013.07.101.
[51] T. Mosmann, Rapid colorimetric assay for cellular growth and survival: application
to proliferation and cytotoxicity assays, J. Immunol. Methods 65 (1983) 55–63,
https://doi.org/10.1016/0022-1759(83)90303-4.
[52] J.M. Thomas, K.M. Bryson, J.L. Meier, Nucleotide Resolution Sequencing of N4-
acetylcytidine in RNA, 1st ed., Elsevier Inc., 2019 https://doi.org/10.1016/bs.
mie.2019.02.022.
[53] A.P.S. Bertoni, R.P. de Campos, A.S.K. Tamajusuku, G.P. Stefani, E. Braganhol, A.
M.O. Battastini, M.R. Wink, Biochemical analysis of ectonucleotidases on primary
rat vascular smooth muscle cells and in silico investigation of their role in vascular
diseases, Life Sci. 256 (2020), 117862, https://doi.org/10.1016/j.lfs.2020.117862.
[54] N. Biswas, S. Khanra, A. Sarkar, S. Bhattacharjee, D. Prasad Mandal, A. Chaudhuri,
S. Chakraborty, C. Roy Choudhury, One new azido bridged dinuclear copper(II)
thiosemicarbazide complex: synthesis, DNA/protein binding, molecular docking
study and cytotoxicity activity, New J. Chem. 41 (2017) 12996–13011, https://
doi.org/10.1039/c7nj01998j.
[55] C. Wei, J. Cheng, B. Zhou, L. Zhu, M.A. Khan, T. He, S. Zhou, J. He, X. Lu, H. Chen,
D. Zhang, Y. Zhao, J. Fu, Tripartite motif containing 28 (TRIM28) promotes breast
cancer metastasis by stabilizing TWIST1 protein, Sci. Rep. 6 (2016) 1–12, https://
doi.org/10.1038/srep29822.
[
[
[
[
[
[
[
27] E.S. Hickman, M.C. Moroni, K. Helin, The role of p53 and pRB in apoptosis and
cancer, Curr. Opin. Genet. Dev. 12 (2002) 60–66, https://doi.org/10.1016/S0959-
4
37X(01)00265-9.
28] E. Siddiqui, I. Azad, D.A. Khan, D.T. Khan, Thiosemicarbazone complexes as
versatile medicinal chemistry agents: a review, J. Drug Deliv. Ther. 9 (2019)
6
89–703, https://doi.org/10.22270/jddt.v9i3.2888.
29] R. Tada, N. Chavda, M.K. Shah, Synthesis and characterization of some new
thiosemicarbazide derivatives and their transition metal complexes, J. Chem.
Pharm. Res. 3 (2011) 290–297.
30] N. Rajendran, A. Periyasamy, N. Kamatchi, V. Solomon, Biological evaluation of
copper(II) complexes on N(4)ꢀ substituted thiosemicarbazide derivatives and
diimine co-ligands using DNA interaction, antibacterial and in vitro cytotoxicity,
J. Coord. Chem. 72 (2019) 1937–1956, https://doi.org/10.1080/
0
0958972.2019.1634806.
[
[
31] J. Devi, M. Yadav, D.K. Jindal, D. Kumar, Y. Poornachandra, Synthesis,
spectroscopic characterization, biological screening and in vitro cytotoxic studies
of 4-methyl-3-thiosemicarbazone derived Schiff bases and their Co (II), Ni (II), Cu
(
II) and Zn (II) complexes, Appl. Organomet. Chem. 33 (2019) 1–23, https://doi.
org/10.1002/aoc.5154.
32] K. Gałczy n´ ska, K. Ciepluch, Ł. Madej, K. Kurdziel, B. Maciejewska, Z. Drulis-Kawa,
A. Węgierek-Ciuk, A. Lankoff, M. Arabski, Selective cytotoxicity and antifungal
properties of copper(II) and cobalt(II) complexes with imidazole-4-acetate anion or
1
4
-allylimidazole, Sci. Rep. 9 (2019) 1–13, https://doi.org/10.1038/s41598-019-
6224-6.
[
[
33] P. Yu, J. Deng, J. Cai, Z. Zhang, J. Zhang, M. Hamid Khan, H. Liang, F. Yang,
Anticancer and biological properties of a Zn-2,6-diacetylpyridine bis
(
1
thiosemicarbazone) complex, Metallomics 11 (2019) 1372–1386, https://doi.org/
0.1039/c9mt00124g.
34] F. Bisceglie, N. Orsoni, M. Pioli, B. Bonati, P. Tarasconi, C. Rivetti, D. Amidani,
S. Montalbano, A. Buschini, G. Pelosi, Cytotoxic activity of copper(ii), nickel(ii)
and platinum(ii) thiosemicarbazone derivatives: interaction with DNA and the H2A
histone peptide, Metallomics 11 (2019) 1729–1742, https://doi.org/10.1039/
c9mt00166b.
35] O. Palamarciuc, M.N.M. Milunovi ´c , A. Sîrbu, E. Stratulat, A. Pui, N. Gligorijevic,
S. Radulovic, J. Ko ˇz í ˇs ek, D. Darvasiov a´ , P. Rapta, E.A. Enyedy, G. Novitchi,
S. Shova, V.B. Arion, Investigation of the cytotoxic potential of methyl imidazole-
derived thiosemicarbazones and their copper(ii) complexes with dichloroacetate as
a co-ligand, New J. Chem. 43 (2019) 1340–1357, https://doi.org/10.1039/
c8nj04041a.
[56] D. Schneidman-Duhovny, Y. Inbar, R. Nussinov, H.J. Wolfson, PatchDock and
SymmDock: servers for rigid and symmetric docking, Nucleic Acids Res. 33 (2005)
363–367, https://doi.org/10.1093/nar/gki481.
[57] M.S. Singh, M.D. Raju, A.K. Singh, P. Narayan, R.J. Rao, Some complexes of
organotin(IV) chloride with salicylaldehyde thiosemicarbazone, Synth. React.
Inorg. Met. Chem. 29 (1999) 73–85, https://doi.org/10.1080/
[
00945719909349435.
[58] T.S. Lobana, S. Khanna, G. Hundal, P. Kaur, B. Thakur, S. Attri, R.J. Butcher,
Coinage metal derivatives of salicylaldehyde thiosemicarbazones: synthesis,
structures, bond isomerism and H-bonded networks, Polyhedron 28 (2009)
1583–1593, https://doi.org/10.1016/j.poly.2009.03.019.
[
[
36] H. Kaur, V.K. Gupta, M. Gupta, Synthesis and Anti Proliferative Activity of
Thiosemicarbazone and 4-Thiazolidinones, 2019, pp. 2–10, https://doi.org/
¨
[59] T. Bal-Demirci, G. Congur, A. Erdem, S. Erdem-Kuruca, N. Ozdemir, K. Akgün-Dar,
1
0.23880/ipcm-16000166.
B. Varol, B. Ülküseven, Iron(III) and nickel(ii) complexes as potential anticancer
agents: synthesis, physicochemical and structural properties, cytotoxic activity and
DNA interactions, New J. Chem. 39 (2015) 5643–5653, https://doi.org/10.1039/
c5nj00594a.
37] M. Muralisankar, R. Dheepika, J. Haribabu, C. Balachandran, S. Aoki, N.S.
P. Bhuvanesh, S. Nagarajan, Design, synthesis, DNA/HSA binding, and cytotoxic
activity of half-sandwich Ru(II)-arene complexes containing
triarylamine–thiosemicarbazone hybrids, ACS Omega 4 (2019) 11712–11723,
https://doi.org/10.1021/acsomega.9b01022.
[60] Z. Afrasiabi, E. Sinn, J. Chen, Y. Ma, A.L. Rheingold, L.N. Zakharov, N. Rath,
S. Padhye, Appended 1,2-naphthoquinones as anticancer agents 1: synthesis,
structural, spectral and antitumor activities of ortho-naphthaquinone
thiosemicarbazone and its transition metal complexes, Inorg. Chim. Acta 357
(2004) 271–278, https://doi.org/10.1016/S0020-1693(03)00484-5.
[61] B. Atasever, B. Ülküseven, T. Bal-Demirci, S. Erdem-Kuruca, Z. Solakoǧlu,
Cytotoxic activities of new iron(III) and nickel(II) chelates of some S-methyl-
thiosemicarbazones on K562 and ECV304 cells, Investig. New Drugs 28 (2010)
421–432, https://doi.org/10.1007/s10637-009-9272-2.
[
[
38] A.R. Aguirre, G.L. Parrilha, R. Diniz, B.C. Ribeiro, R.G.D. Santos, H. Beraldo,
Cytotoxic effects of indium(III) complexes with 2-acetylpyridine-N(4)-ortho-fluo-
rophenylthiosemicarbazone and their radioactive 114mIn analogues against
human glioma cells, Polyhedron 164 (2019) 219–227, https://doi.org/10.1016/j.
poly.2019.02.055.
39] D. Polo-Cer o´ n, Cu(II) and Ni(II) complexes with new tridentate NNS
thiosemicarbazones: synthesis, characterisation, DNA interaction, and antibacterial
activity, Bioinorg. Chem. Appl. 2019 (2019), https://doi.org/10.1155/2019/
[62] A. Matuszewska, M. Jaszek, D. Stefaniuk, T. Ciszewski, Ł. Matuszewski,
Anticancer, antioxidant, and antibacterial activities of low molecular weight
bioactive subfractions isolated from cultures of wood degrading fungus Cerrena
unicolor, PLoS One 13 (2018) 1–14, https://doi.org/10.1371/journal.
pone.0197044.
[63] R. Roy, J. Chun, S.N. Powell, BRCA1 and BRCA2: different roles in a common
pathway of genome protection, Nat. Rev. Cancer 12 (2012) 68–78, https://doi.org/
10.1038/nrc3181.
3
520837.
[
[
[
40] M. Bhattacharya, S.A. Iqbal, S. Malik, Spectral and diuretic study of Cu(II) complex
of sulfonamides, Der Chem. Sin. 3 (2012) 1204–1212. http://www.imedpub.
com/articles/spectral-and-diuretic-study-of-cuii-complex-of-sulfonamides.pdf.
41] V. Kalapala, V. Banothu, S.K.B. Chandra, K. Mukkantia, Synthesis and
characterization of some new thiosemicarbazide derivatives and their transition
metal complexes, J. Chem. Pharm. Res. 7 (2015) 437–445.
42] M. Belicchi Ferrari, F. Bisceglie, G. Pelosi, M. Sassi, P. Tarasconi, M. Cornia,
S. Capacchi, R. Albertini, S. Pinelli, Synthesis, characterization and X-ray structures
of new antiproliferative and proapoptotic natural aldehyde thiosemicarbazones
[64] T. Shahid, J. Soroka, E.H. Kong, L. Malivert, M.J. McIlwraith, T. Pape, S.C. West,
X. Zhang, Structure and mechanism of action of the BRCA2 breast cancer tumor
1
3

B.Z. Sibuh et al.
Life Sciences 273 (2021) 119305
suppressor, Nat. Struct. Mol. Biol. 21 (2014) 962–968, https://doi.org/10.1038/
genomic instability in lung cancer, Int. J. Genomics. 2019 (2019), https://doi.org/
10.1155/2019/9584504.
nsmb.2899.
[
[
[
65] N. Petrucelli, M.B. Daly, G.L. Feldman, Hereditary breast and ovarian cancer due to
mutations in BRCA1 and BRCA2, Genet. Med. 12 (2010) 245–259, https://doi.org/
[74] E.S. Wenzel, A.T.K. Singh, Cell-cycle checkpoints and aneuploidy on the path to
cancer, In Vivo (Brooklyn) 32 (2018) 1–5, https://doi.org/10.21873/
invivo.11197.
[75] R. Spencer-Smith, J.P. O’Bryan, Direct inhibition of RAS: quest for the holy grail?
Semin. Cancer Biol. 54 (2019) 138–148, https://doi.org/10.1016/j.
semcancer.2017.12.005.
[76] T.E. Mattox, X. Chen, Y.Y. Maxuitenko, A.B. Keeton, G.A. Piazza, Exploiting RAS
nucleotide cycling as a strategy for drugging RAS-driven cancers, Int. J. Mol. Sci.
21 (2020), https://doi.org/10.3390/ijms21010141.
1
0.1097/GIM.0b013e3181d38f2f.
66] L.Y. Marmorstein, T. Ouchi, S.A. Aaronson, The BRCA2 gene product functionally
interacts with p53 and RAD51, Proc. Natl. Acad. Sci. U. S. A. 95 (1998)
1
3869–13874, https://doi.org/10.1073/pnas.95.23.13869.
67] S. Rajagopalan, A. Andreeva, T.J. Rutherford, A.R. Fersht, Mapping the physical
and functional interactions between the tumor suppressors p53 and BRCA2, Proc.
Natl. Acad. Sci. U. S. A. 107 (2010) 8587–8592, https://doi.org/10.1073/
pnas.1003689107.
[77] X. Zhang, Z. Qi, H. Yin, G. Yang, Interaction between p53 and ras signaling controls
[
[
68] L. Raimundo, H. Ramos, J.B. Loureiro, J. Calheiros, L. Saraiva, BRCA1/P53: two
strengths in cancer chemoprevention, Biochim. Biophys. Acta - Rev. Cancer. 1873
cisplatin resistance via HDAC4- and HIF-1 -mediated regulation of apoptosis and
autophagy, Theranostics 9 (2019) 1096–1114, https://doi.org/10.7150/
thno.29673.
α
(
2020), 188339, https://doi.org/10.1016/j.bbcan.2020.188339.
69] Y.L. Chai, J.Q. Cui, N. Shao, E.S.P. Reddy, V.N. Rao, The second BRCT domain of
BRCA1 proteins interacts with p53 and stimulates transcription from the p21
[78] B.L. Allen-Petersen, R.C. Sears, Mission possible: advances in MYC therapeutic
targeting in cancer, BioDrugs 33 (2019) 539–553, https://doi.org/10.1007/
s40259-019-00370-5.
[79] P.E. Tokgun, O. Tokgun, S. Kurt, A.G. Tomatir, H. Akca, MYC-driven regulation of
long non-coding RNA profiles in breast cancer cells, Gene 714 (2019), 143955,
https://doi.org/10.1016/j.gene.2019.143955.
[80] A. Santoro, T. Vlachou, L. Luzi, G. Melloni, L. Mazzarella, E. D’Elia, X. Aobuli, C.
E. Pasi, L. Reavie, P. Bonetti, S. Punzi, L. Casoli, A. Sab o` , M.C. Moroni, G.I. Dellino,
B. Amati, F. Nicassio, L. Lanfrancone, P.G. Pelicci, p53 loss in breast cancer leads to
Myc activation, increased cell plasticity, and expression of a mitotic signature with
prognostic value, Cell Rep. 26 (2019) 624–638.e8, https://doi.org/10.1016/j.
celrep.2018.12.071.
[81] K.G. Ponder, L.H. Boise, The prodomain of caspase-3 regulates its own removal and
caspase activation, Cell Death Dis. 5 (2019) 1–10, https://doi.org/10.1038/
s41420-019-0142-1.
(
WAF1/CIP1) promoter, Oncogene 18 (1999) 263–268, https://doi.org/10.1038/
sj.onc.1202323.
[
70] X. Xu, W. Qiao, S.P. Linke, L. Cao, W.M. Li, P.A. Furth, C.C. Harris, C.X. Deng,
Genetic interactions between tumor suppressors Brca1 and p53 in apoptosis, cell
cycle and tumorigenesis, Nat. Genet. 28 (2001) 266–271, https://doi.org/10.1038/
9
0108.
[
[
[
71] T.K. MacLachlan, B.C. Dash, D.T. Dicker, W.S. El-Deiry, Repression of BRCA1
through a feedback loop involving p53, J. Biol. Chem. 275 (2000) 31869–31875,
https://doi.org/10.1074/jbc.M003338200.
72] J.D. Oliner, A.Y. Saiki, S. Caenepeel, The role of MDM2 amplification and
overexpression in tumorigenesis, Cold Spring Harb. Perspect. Med. 6 (2016) 1–15,
https://doi.org/10.1101/cshperspect.a026336.
73] G. Soca-Chafre, A. Montiel-D a´ valos, I.A. De La Rosa-Vel a´ zquez, C.H.S. Caro-
S a´ nchez, A. Pe n˜ a-Nieves, O. Arrieta, Multiple molecular targets associated with
1
4