Ahmed A Noser1, Ihsan A Shehadi2, Aboubakr H Abdelmonsef3, Maha M Salem4. 1. Organic Chemistry, Chemistry Department, Faculty of Science, Tanta University, Tanta 31527, Egypt. 2. Department of Chemistry, Pure and Applied Chemistry Research Group, College of Sciences, University of Sharjah, Sharjah 27272, UAE. 3. Chemistry Department, Faculty of Science, South Valley University, Qena 83523, Egypt. 4. Biochemistry Division, Chemistry Department, Faculty of Science, Tanta University, Tanta 31527, Egypt.
Abstract
A series of novel pyrazolinone chalcones 3-9 have been synthesized through the condensation of azo pyrazolinone derivatives with various aromatic aldehydes. Spectroscopic techniques and elemental analysis have both corroborated this. Furthermore, all compounds were screened in silico for their ability to inhibit cancer proliferation and metastasis by targeting the PI3K/Akt signaling pathway. This inhibitory pathway might be an efficient approach for the death of cancer cells, angiogenesis, and metastasis prevention. Our results indicated that only compound 6b was the top-ranked. It demonstrated the highest binding energies of -11.1 and -10.7 kcal/mol against the target proteins PI3K and Akt, respectively; thus, it was chosen for in vitro studies. Compound 6b exhibited the most effective cytotoxic impact against the Caco cell line with IC50 of 23.34 ± 0.14 μM. Furthermore, it showed significant inhibition of PI3K/Akt proteins and oxidative stress, leading to elevated Bax and p53 expression, reduced Bcl-2 expression, and triggered cell cycle arrest at the sub-G0/G1 phase. Additionally, it showed significant downregulation of the Raf-1 gene, leading to ERK1/2 protein inhibition. These findings demonstrate that compound 6b obeyed Lipinski's rule of five and might be used as a favored scaffold for cancer treatment by inhibiting proliferation and metastasis via inhibition of the PI3K/Akt and Raf-1/ERK1/2 signaling pathways.
A series of novel pyrazolinone chalcones 3-9 have been synthesized through the condensation of azo pyrazolinone derivatives with various aromatic aldehydes. Spectroscopic techniques and elemental analysis have both corroborated this. Furthermore, all compounds were screened in silico for their ability to inhibit cancer proliferation and metastasis by targeting the PI3K/Akt signaling pathway. This inhibitory pathway might be an efficient approach for the death of cancer cells, angiogenesis, and metastasis prevention. Our results indicated that only compound 6b was the top-ranked. It demonstrated the highest binding energies of -11.1 and -10.7 kcal/mol against the target proteins PI3K and Akt, respectively; thus, it was chosen for in vitro studies. Compound 6b exhibited the most effective cytotoxic impact against the Caco cell line with IC50 of 23.34 ± 0.14 μM. Furthermore, it showed significant inhibition of PI3K/Akt proteins and oxidative stress, leading to elevated Bax and p53 expression, reduced Bcl-2 expression, and triggered cell cycle arrest at the sub-G0/G1 phase. Additionally, it showed significant downregulation of the Raf-1 gene, leading to ERK1/2 protein inhibition. These findings demonstrate that compound 6b obeyed Lipinski's rule of five and might be used as a favored scaffold for cancer treatment by inhibiting proliferation and metastasis via inhibition of the PI3K/Akt and Raf-1/ERK1/2 signaling pathways.
Cancer is a potentially
fatal disease that affects people worldwide.
The environmental variables account for 90–95% of all cancer
cases, with the remaining cases 5–10% attributed to inherited
genetic factors. Moreover, surgery, radiation, and chemotherapy were
the mainstays of cancer treatment in the past. Even though chemotherapy
is the most often used treatment, several chemotherapeutic drugs have
harmful side effects, including the indiscriminate death of normal
cells and chemotherapeutic resistance.[1] As a result, a constant quest for alternative, targeted, novel,
efficacious, and less toxic anticancer agents that inhibit metabolic
target proteins are urgently needed. Therefore, designing the bioenergetic
drugs that disrupt the metabolic route essential for cancer cell survival,
metastasis, and proliferation is of critical importance in this context.[2]Akt is a proto-oncogenic serine–threonine
kinase that plays
a role in cell proliferation, glucose metabolism, cell migration,
and apoptosis. Akt is activated in tumor cells via phosphorylation,
which arises when a PI3K-phosphorylated phosphoinositide (PI) termed
PIP3 attaches to the homology Akt domain, followed by translocation
to the plasma membrane and phosphorylation by PDK1 and PDK2 at two
phosphorylation Ser473 and Thr308 sites. As a result, direct PI3K/Akt
degradation was discovered to inhibit Akt activity and induce apoptosis.[3] For several years, inhibition of Akt has been
regarded as a promising therapeutic approach in oncology as it leads
to the inhibition of Raf-1, which inhibits the proliferating and angiogenesis
proteins, MEK and ERK1/2. Moreover, oxidative stress inhibits the
PI3K/Akt signaling pathway and promotes reactive oxygen species (ROS)
generation, resulting in Bax and p53 activation, cell cycle arrest,
and cancer cell apoptosis.[4] Many efforts
have been undertaken to design new anticancer agents that are both
selective and effective to inhibit PI3K/Akt and Raf-1/ERK1/2 as they
can worsen the proliferation of cancer cells, leading to global programming
of cancer cell death and inducing mitochondrial dysfunction because
of oxidative stress[5,6] (Figure ).
Figure 1
Schematic diagram of the design strategy signaling
pathways.
Schematic diagram of the design strategy signaling
pathways.Chalcones possess many biological
activities, including antibacterial,
antifungal, antimalarial, anticonvulsant, and anticancer agents.[7] Furthermore, these compounds are of high interest
because of their use in synthesizing many biologically active heterocycles
such as azepines, pyrazolines, and flavones.[8−11]Pyrazolinones have a wide
range of biological effects, including
anti-inflammatory, antibacterial, antifungal, analgesic, antidiabetic,
antioxidant, and anticancer properties. One of the most effective
drug-bearing pyrazoline moieties is Axitinib, which is used as an
anticancer drug. To create effective anticancer medicines, pyrazoline
is also hybridized with other nitrogen-, sulfur-, and oxygen-containing
heterocyclic scaffolds such as quinoline, indole, oxazole, and thiazole.[12−14]This project aimed to design and synthesize novel anticancer
scaffolds
of pyrazolinone chalcones due to the importance of pyrazolinones and
chalcones in the medical area. Computer-based docking experiments
were performed on the produced compounds to study the binding mechanism
with the active sites of the target enzymes. In addition, in silico
physicochemical and pharmacokinetic properties were carried out to
estimate the absorption, distribution, metabolism, excretion, and
toxicity (ADMET) properties of the compounds as well as investigate
the structure–activity relationship (SAR). Ultimately, the
chosen compound was investigated in vitro to confirm its inhibitory
efficacy toward cancer cell proliferation and metastasis by targeting
PI3K/Akt and Raf-1/ERK1/2 signaling pathway inhibition.
Materials and
Methods
Chemicals and Drugs
Ethyl acetoacetate (EAA), phenylhydrazine,
2,4-dinitrophenylhydrazine (DNP), hydrazine hydrate, p-aminoacetophenone, sodium nitrite, benzaldehyde, 3,5-dimethoxybenzaldehyde,
4-(N,N-dimethylamino)benzaldehyde,
4-hydroxybenzaldehyde, 2-nitrobenzaldehyde, cinnamaldehyde, 4-(N,N-dimethylamino)cinnamaldehyde, sodium
hydroxide, ethyl alcohol, glacial acetic acid, trichloroacetic acid
(TCA), sodium pyrophosphate, reduced glutathione (GSH), and thiobarbituric
acid (TBA) were obtained from Sigma-Aldrich Chemical Co. (St. Louis,
MO, USA). Doxorubicin HCl injection, USP, was purchased from Pfizer
injectables.
General Information
Thin-layer chromatography monitored
the reactions performed on precoated Merck Kieselgel 60 F254 plates
(EMD Millipore Corporation, Billerica, MA, USA). A PerkinElmer 1420
spectrophotometer (Waltham, MA, USA) was used to record infrared spectra
at the Central Laboratory of Tanta University. The KBr disc technique
was used to obtain the spectra. The samples were mounted on a sample
holder with a big cavity after drying. The open capillary method was
used to estimate melting points, which were calculated using the Gallenkamp
melting point and reported uncorrected. Electron impact mass spectrometry
(EIMS) was used to measure mass spectra at 70 eV at Al-Azhar University’s
Regional Center for Mycology and Biotechnology. A PerkinElmer 240
CHN Elemental Analyzer was used to undertake elemental analysis of
substances at Cairo University’s Microanalytical Center. The 1H NMR spectra were recorded on a Bruker AC spectrometer (400
MHz) and 13C NMR (100 MHz at 25 °C in DMSO-d6 with tetramethylsilane as an internal standard.
The chemical shifts for 1H NMR are reported in ppm from
tetramethylsilane (0 ppm) or referenced to the solvent (DMSO-d6, δ2.50). Chemical shifts (δ) for 13C NMR spectra are referenced to the signals for residual
deuterated solvents (DMSO-d6, 37.5). Multiplicities
are reported by the following abbreviations: s (singlet), d (doublet),
m (multiplet).
Synthesis of Pyrazolinone Derivatives (1a–c)
A mixture of EAA (10 mmol, 1.28 mL)
and hydrazine derivatives
(10 mmol) in 30 mL of ethanol was refluxed for 18 h, then cooled,
filtered, and recrystallized from ethanol to give compounds 1a–c as described by Alharthy.[15]
Synthesis of Azo Pyrazolinone Derivatives (2a–c)
Azo pyrazolinone derivatives (2a–c) were prepared by coupling the pyrazolinone (1a–c) with a freshly prepared solution of p-acetyl phenyl
diazoniumchloride in the presence of aluminum chloride (AlCl3) as described by Khalil.[16]
General Procedure for the
Synthesis of Pyrazolinone Chalcones
(3–9)
In a 50 mL conical flask, a mixture
of aromatic aldehyde (1 mmol), azo pyrazolinone derivatives (2a–c, 1 mmol), sodium hydroxide (0.08 g, 2 mmol), and
ethanol (10.0 mL) was stirred at room temperature for 20 h (TLC control,
petroleum ether/ethyl acetate: 8:2). Then the reaction mixture was
poured into ice water, filtered off, and dried.
In the current research, molecular
docking studies[17−24] were performed to explore the binding modes of the ligand molecules
toward the target proteins PI3K and Akt. The crystal structures of
the targets were retrieved from the RCSB protein data bank.[25] The target files were optimized by removing
the co-crystalized ligands, heteroatoms, and water molecules. In addition,
their energies were minimized using CHARM Force Field[26] in Discovery Studio 3.5 Visualizer. Further, the 2D structures
of the prepared analogues were generated in cdx format (2D structures)
using ChemDraw Ultra 8.0 and then converted to sdf files (3D structures)
using the Open Babel GUI 2.4.1 tool.[27] Furthermore,
the UFF force field[28] in the PyRx tool
was used to minimize their energies. An in-house library of 12 ligand
molecules was generated for the docking. The in silico docking technique
was performed using PyRx—a virtual screening tool.[29] Grid maps of 25 × 25 × 25 Å3 were generated around the active site region of the target
proteins, resulting in nine conformers for each docked molecule, and
the minimum binding energy was selected for further study. The 2D
and 3D representations of docking results were visualized using Discovery
Studio 3.5. Finally, the drug-like properties of the newly prepared
molecules were calculated using mol inspiration, Swiss ADME, and Admet
SAR web tools.
Anticancer Investigations on the Expected
Compound (In Vitro)
The anticancer impact was studied using
the tetrazolium 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium
bromide (MTT) assay on the most effective compound resulting from
the in silico studies and then submitted to further analyses.
Cell Line
Maintenance and Treatment
The lung cancer
cell line (A549), triple-negative breast cancer cell line (MDA-231),
pancreatic cancer cell line (PCL), estrogen receptor-positive breast
cancer cell line (MCF-7), colon cancer cell line (Caco), and WISH
normal cell line were seeded with (1 × 104 cells/well)
separately using complete media containing (Dulbecco’s modified
Eagle’s medium with 10% fetal bovine serum and 1% penicillin/streptomycin)
in a 5% CO2 incubator and a 95% humidified environment
at 37 °C. All cell lines were provided by the Center of Excellence
for Research in Regenerative Medicine and Its Applications, Alexandria
University, Egypt. The cell lines were incubated with the selected
compound at several concentrations (0–200 μM) and doxorubicin
(DOX) as a standard chemotherapeutic drug (0–100 μM)
for 48 h, and then, the viability of the cells was determined using
the MTT assay (Gibco-BRL, New York, NY, USA).[30,31]
Cell Morphology Study
Briefly, 1 × 105 of the Caco cell line was seeded in a six-well plate, incubated
for 24 h, and then treated dose-dependently with 1/4IC50, 1/2IC50, and IC50 of the selected compound.
After 48 h incubation, morphological alterations of treated and untreated
cells were evaluated and captured using an inverted light microscope
(Olympus, USA).
Cell Cycle Examination
Flow cytometry
was used to analyze
cell cycle phases using an Accuri C6 flow cytometer (Becton Dickinson
BD, USA) on Caco cells 1 × 105 that were trypsinized,
centrifuged at 5000 rpm, then washed with 1× cold phosphate-buffered
saline (PBS), and fixed with cold absolute ethanol as described by
Noser et al. and Darzynkiewicz et al.[31,32]
qPCR Assessment
The Caco 1 × 105 control
and treated cells were trypsinized, centrifuged at 4500 rpm, and washed
with 1× PBS. Then the pelleted Caco cells were subjected to RNA
extraction and transcription to cDNA as described by Kvastad et al.[33] The expression of Raf-1, p53, Bax, and Bcl–2 mRNA was measured using Applied qPCR Biosystems (Foster
City, USA) on treated and control Caco cells according to Livak and
Schmittgen.[34] The primer sequences were
designed using primer 3plus as in Table .
Table 1
Primer Sequences
Used in qRT-PCR
gene
forward primer (/5---/3)
reverse primer (/5---/3)
Raf-1
GCAGGATAACAACCCATTC
GGTCAGCGTGCAAGCATT
P53
TAACAGTTCCTGCATGGGCGGC
AGGACAGGCACAAACACGCACC
Bax
GGCTGGACACTGGACTTCCT
GGTGAGGACTCCAGCCACAA
Bcl–2
TTCGCAGAGATGTCCAGTCA
TTCAGAGACAGCCAGGAGAA
GAPDH
TGTGTCCGTCGTGGATCTGA
CCTGCTTCACCACCTTCTTGA
Biomarker Estimation of
Antioxidant/Oxidative Stress
The 1 × 105 Caco
cells were treated for 48 h. Subsequently,
the cells were scraped, and pelleted cells were washed twice with
1× cold PBS. The scraped pelleted cells were incubated in the
lysis buffer as described by Noser et al.[31] to measure the levels of malondialdehyde (MDA) and the activity
of reduced glutathione (GSH).[35] The protein
content was determined using Bradford.[36]
Western Blot Analysis
The method of Mruk and Cheng[37] was used for immunoblotting. Proteins are removed
from Caco control and treated cells using cold RIPA lysis buffer and
quantified using Bradford.[36] Equal amounts
of proteins (20 μg) were separated and transferred to a polyvinylidene
difluoride (PVDF) membrane. After blocking the membrane, the primary
antibodies as phospho-PI3K (ab182651), phospho-Akt (ab81283), and
phospho-ERK1/2 (ab214362) were added and incubated with it. Then,
the primary antibodies were removed, carefully washed several times,
and incubated with the secondary antibody horseradish peroxidase (HRP)-conjugated
goat anti-rabbit IgG (H + L) (ab205718). The bands were visualized
and normalized with β-actin as described by Noser et al.[31]
Statistical Analysis
The experimental
results are presented
as mean ± SE. GraphPad Prism 6 software was used to determine
the significance of differences between the control and treated groups
using one and two-way ANOVA.
Results and Discussion
Chemistry
of the Synthesized Compounds
The reaction
of EAA with hydrazine derivatives led to the formation of pyrazolinone
derivatives (1a–c) with high yields as described
by Alharthy.[15]p-Acetyl
phenyl diazonium chloride was coupled with the pyrazolinone derivatives
(1a–c) to give the azo pyrazolinone derivatives
(2a–c) with 92, 91, and 89% yields, respectively,
as described in Scheme .
Scheme 1
Synthesis Pathway of Compounds 1 and 2
The chalcones (3–9) were synthesized from the
reaction of azo pyrazolinone derivatives (2a–c) with different substituted aromatic aldehydes in a basic medium
as described in Scheme .
Scheme 2
Synthesis Pathway of Compounds 3–9
All synthesized compounds 2–9 were characterized
using different spectroscopic techniques and elemental analysis.
In Silico Docking Study
PI3K/Akt is one of the major
signaling pathways associated with tumor proliferation in human cancer.[38] Therefore, these proteins are selected as pivotal
therapeutic targets for identifying cancer agents. In the present
study, the molecular docking approach was achieved to explore new
PI3K/Akt drug candidates. The screened compounds exhibited docking
scores between −8.3 and −11.1 kcal/mol and between −8.6
and −10.7 kcal/mol against PI3K and Akt targets, respectively.
The molecular docking studies resulted in compound 6b having the highest binding energy against the proteins. Figure shows the 2D and
3D representations of the best-docked compound 6b intermolecular
interactions with both targets. Compound 6b docked to
the protein PI3K through five H-bonds and two π-cation interactions
with the amino acid residues Asn114, Val125, Cys126, Glu78, Ser690,
Arg140, and Arg693 at distances of 2.99, 2.98, 2.95, 2.44, 2.71, 5.86,
and 4.17, respectively (Table ). In addition, compound 6b interacted with the
protein Akt through five H-bonds and one π-cation. The SAR study
showed that heterocyclic rings as pyrazolinone and phenolic moieties
in compound 6b play a significant role for enhancing
its activity.
Figure 2
(Left side) 2D and (right side) 3D representations of
interactions
of the best-docked compound 6b with amino acid residues
of the targets PI3K and Akt.
Table 2
Calculated Docking Scores (in kcal/mol)
of the Best-Docked Compound with the Targets
PI3K
Akt
compounds
docking score (ΔGbind)
docked complex (amino acid–ligand) interactions
distance
(Å)
docking score (ΔGbind)
docked complex (amino acid–ligand) interactions
distance
(Å)
6b
–11.1
H-bond
–10.7
H-bond
Asn114---compound 6b
2.99
Thr195:OG1---compound 6b
2.12
Val125---compound 6b
2.98
Thr195:OG1---compound 6b
2.98
Cys126---compound 6b
2.95
Thr195:OG1---compound 6b
2.84
Glu78:OE1---compound 6b
2.44
Ala230:N---compound 6b
2.79
Ser690:OG---compound 6b
2.71
Glu228:O---compound 6b
2.35
π-cation
π-cation
Arg140:NH1---compound 6b
5.86
Lys179:NZ---compound 6b
4.20
Arg693:NH2---compound 6b
4.17
(Left side) 2D and (right side) 3D representations of
interactions
of the best-docked compound 6b with amino acid residues
of the targets PI3K and Akt.Moreover,
we performed further analysis for the compounds such
as ADMET and drug-like properties, as represented in Table . The results suggested that
the intestinal barrier may well absorb all compounds but not at the
blood–brain barrier level. In addition, all compounds except 4c, 5c, 6c, 7c, 8c, and 9c obeyed the Lipinski rule of five (Ro5)
by not having over one violation.
Table 3
ADMET and Drug-likeness
Profiles of
the Compounds
molecular
weight (g/mol)
blood–brain barrier (BBB+)
Caco-2 permeability
(Caco2+)
% human intestinal absorption (HIA+)
TPSA A2
logp
HBA
HBD
N rotatable
N violations
GI absorption
carcinogenicity
acceptable ranges
130–500
–3 to 1.2
<25 poor, 500 great
<80% high, >25% low
≤140
<5
2.0–20.0
0.0–6.0
≤10
≤1
noncarcinogenic
3a
332.36
0.97
55.68
99.53
83.25
1.78
5
1
5
0
high
noncarcinogenic
3b
408.45
0.98
61.78
100.00
74.46
3.15
5
0
6
0
high
noncarcinogenic
3c
498.45
0.90
50.86
99.39
166.12
1.47
9
0
8
1
low
noncarcinogenic
4a
392.41
0.83
51.72
98.24
101.71
1.18
7
1
7
0
high
noncarcinogenic
4b
468.50
0.87
53.48
99.66
92.92
2.50
7
0
8
0
high
noncarcinogenic
4c
558.50
0.80
50.00
99.26
184.66
0.92
11
0
10
2
low
noncarcinogenic
5a
375.42
0.88
57.66
99.13
86.49
1.70
5
1
6
0
high
noncarcinogenic
5b
451.52
0.94
62.83
100.00
77.70
3.02
5
0
7
0
high
noncarcinogenic
5c
541.51
0.74
51.45
99.29
169.36
1.39
9
0
9
2
low
noncarcinogenic
6a
348.36
0.79
55.60
98.95
103.48
1.25
6
2
5
0
high
noncarcinogenic
6b
424.45
0.89
53.85
100.00
94.69
2.61
6
1
6
0
high
noncarcinogenic
6c
514.45
0.73
52.45
99.13
186.35
0.99
10
1
8
2
high
noncarcinogenic
7a
377.35
0.85
50.75
95.12
129.07
0.90
7
1
6
0
high
noncarcinogenic
7b
453.45
0.90
50.86
99.39
120.28
2.27
7
0
7
0
high
noncarcinogenic
7c
543.44
0.90
50.86
99.39
211.94
0.72
11
0
9
2
low
noncarcinogenic
8a
358.39
0.97
55.68
99.53
83.25
2.16
5
1
6
0
high
noncarcinogenic
8b
434.49
0.98
61.78
100.00
74.46
3.48
5
0
7
0
high
noncarcinogenic
8c
524.48
0.90
50.86
99.31
166.12
1.81
9
0
9
2
low
noncarcinogenic
9a
401.46
0.88
57.66
99.13
86.49
2.07
5
1
7
0
high
noncarcinogenic
9b
477.56
0.94
62.83
100.00
77.70
3.35
5
0
8
0
high
noncarcinogenic
9c
567.55
0.74
51.45
99.29
169.36
1.72
9
0
10
2
low
noncarcinogenic
Conversely, the bioavailability radar (Figure ) of the best-docked molecule 6b showed that the pink-colored zone is the perfect space for each
property. The plot suggested that compound 6b could be
a potential new anticancer drug candidate.
Figure 3
Oral bioavailability
radar of the best compound 6b.
Oral bioavailability
radar of the best compound 6b.
Antitumor/Cytotoxic (In Vitro) Studies
Compound 6b was chosen from the molecular docking studies to investigate
its potential as a new anticancer drug via inhibiting the PI3K/Akt
and Raf-1/ERK1/2 signaling pathways. Compound 6b showed
significant antitumor effects on A549, MDA-231, Caco, PCL, and MCF-7
cancer cell lines with IC50 values equal to 40.91 ±
0.35, 38.45 ± 0.29, 23.34 ± 0.14, 56.33 ± 0.22, and
50.15 ± 0.14 μM, respectively. Our results showed that
compound 6b had more significant inhibitory effects on
the Caco colon cancer cell line compared with DOX as a reference drug
(IC50 = 6.713 ± 0.27 μM). Moreover, compound 6b showed lower cytotoxic effects on WISH normal cells (IC50 = 124.4 ± 1.7 μM). This means that it is more
powerful against cancer cell proliferation while having no damaging
side effects on healthy cells; this is in line with the in silico
results. DOX, on the other hand, has an extremely cytotoxic effect
on normal cells (IC50 = 19.27 ± 0.31 μM) (Figure ). Thus, the Caco
cell line was selected for further analysis.
Figure 4
Antitumor/cytotoxic effect
of compound 6b and doxorubicin
on various cell lines.
Antitumor/cytotoxic effect
of compound 6b and doxorubicin
on various cell lines.
Alternation in Morphological
Features
Cytotoxic agents
infrequently alter the cell morphology, resulting in an abnormal cell
morphology, increased cell debris, and decreased cell numbers. In
the present study, detectable morphological features of apoptosis
were observed in Caco cells treated with compound 6b in
a dose-dependent manner (1/4IC50, 1/2IC50, IC50), including cellular shrinkage with the cell number reduced
and detachment, cell rounding, and cytoplasmic condensation. However,
the morphology of the untreated cells appeared normal and confluent,
as shown in Figure . These results further elaborated the ability of compound 6b in inducing apoptosis.
Figure 5
Morphological features of apoptosis in
the Caco cells treated with
compound 6b in a dose-dependent manner (1/4IC50, 1/2IC50, IC50) after 48 h treatment.
Morphological features of apoptosis in
the Caco cells treated with
compound 6b in a dose-dependent manner (1/4IC50, 1/2IC50, IC50) after 48 h treatment.
Cell Cycle Arrest Detection
The
inhibition of the PI3K/Akt
protein kinases causes cell cycle arrest. Moreover, this arrest confirmed
the reduction in proliferation and metastasis of the Caco cancer cell
line via inhibiting the Raf-1/ERK1/2 signaling pathway. Compound 6b enhanced the percentage of cells in the sub-G0/G1 phase
in Caco cells at all doses when compared with the untreated cells
(this is the phase in which cells wait to enter the cell cycle to
duplicate; when the number of cells in this phase rises, the cell
cycle has been stopped, and division and replication are impossible).
The 1/4IC50, 1/2IC50, and IC50 of
compound 6b showed cell cycle arrest at rates of 8.8,
17.4, and 30.4%, respectively, in the sub-G0/G1 phase compared to untreated Caco cells (2.2%). This means that compound 6b inhibits cell growth, arrest in cell cycle
progression, and the increase of cells in G1 reflecting its apoptotic
effect, and the G2/M checkpoint blocks the entry into mitosis when
DNA is damaged, as illustrated in Figure . Collectively, these results demonstrated
that compound 6b can induce inhibition to PI3K/Akt and
Raf-1/ERK1/2 signaling pathways and trigger apoptosis via arresting
the cell cycle in the sub-G0/G1 phase in a dose-dependent
manner.[38,39]
Figure 6
Cell cycle phases of compound 6b in the Caco cell
line in a dose-dependent manner (1/4IC50, 1/2IC50, IC50) after 48 h treatment.
Cell cycle phases of compound 6b in the Caco cell
line in a dose-dependent manner (1/4IC50, 1/2IC50, IC50) after 48 h treatment.
qRT-PCR Study
qRT-PCR was used to measure the mRNA
expression of the Bax, p53 (apoptotic markers), Bcl–2 (antiapoptotic marker), and Raf-1 (proliferative marker) genes in
the Caco cell line. In cells treated with compound 6b, the expression of Bax and p53 was considerably (p < 0.0001) increased in a dose-dependent manner, with the highest
expression in the IC50 of cells treated with compound 6b compared to untreated cells. Bcl–2 and
Raf-1 gene expressions were dramatically (p <
0.0001) downregulated in compound 6b-treated cells in
a dose-dependent manner, with a little expression in the IC50 of compound 6b-treated cells compared to untreated
cells, as shown in Figure . As a result, the upregulation of Bax expression and downregulation
of Bcl–2 expression indicate that compound 6b causes mitochondrial membrane dysfunction that causes release
of cytochrome C and activation of the caspase cascade that finally
led to apoptosis. In contrast, the upregulation of p53 expression
and downregulation of the expression of the proliferative gene Raf-1
result from the inhibition of PI3K/Akt and lead to inhibit ERK1/2
signaling pathways. This clarifies the ability of compound 6b to inhibit cell proliferation, angiogenesis, and metastasis, leading
to cell cycle arrest and apoptosis induction.[39,40]
Figure 7
Relative
expression of Raf-1, P53, Bax, and Bcl–2 in the
Caco cell line (compound 6b) in a dose-dependent
manner (1/4IC50, 1/2IC50, IC50) after
48 h treatment.
Relative
expression of Raf-1, P53, Bax, and Bcl–2 in the
Caco cell line (compound 6b) in a dose-dependent
manner (1/4IC50, 1/2IC50, IC50) after
48 h treatment.
Antioxidant/Oxidative Stress
Biomarkers
Our results
revealed that the level of MDA that results from the lipid peroxidation
process occurring in Caco cells treated with compound 6b in a dose-dependent manner (1/4IC50, 1/2IC50, IC50) was increased significantly, while the specific
activity of reduced glutathione (GSH) was remarkably diminished as
compared with the untreated Caco cells, as shown in Figure . This implies that compound 6b could promote programming cell death in cancer cells by
generating intracellular ROS and blocking the antioxidant endogenous
enzymes. According to this theory, the high ROS production in cancer
cells causes malfunction of the mitochondrial membrane, inhibits the
PI3K/Akt protein kinase, and leads to the Raf-1/ERK1/2 signaling pathway
inhibition. Finally, apoptosis is induced, and cell survival, proliferation,
and metastasis are arrested.[38,41−43]
Figure 8
MDA
and GSH levels in the Caco cell line (compound 6b) in
a dose-dependent manner (1/4IC50, 1/2IC50, IC50) after 48 h treatment.
MDA
and GSH levels in the Caco cell line (compound 6b) in
a dose-dependent manner (1/4IC50, 1/2IC50, IC50) after 48 h treatment.
Immunoblotting Confirms the Inhibition of the PI3K/Akt/ERK1/2
Signaling Pathway
Our results elucidated that compound 6b induces a remarkable drop in PI3K/Akt protein kinase with
a significant decrease in ERK1/2 in the Caco colon cancer cell line
dose-dependently as compared to untreated cells, Figure . These findings confirmed
compound 6b’s mechanical routes of inhibition
of PI3K, which inhibits PIP3 and promotes ROS intracellular production,
resulting in Akt inhibition via dephosphorylation. When Akt is dephosphorylated,
p53 is activated due to blocking MDM2 protein and the cell cycle is
stopped. Additionally, it inhibits Raf-1, which leads to inhibition
of MEK protein and finally causes ERK1/2 inhibition, arresting cell
proliferation, angiogenesis, and metastasis.[44−48]
Figure 9
Effects of compound 6b on the phosphorylation
of PI3K/Akt/ERK1/2
in Caco cells. The cells were treated with the 1/4IC50,
1/2IC50, and IC50 for 48 h, and the protein
phosphorylation levels were relative to β-actin protein (internal
control) using western blot analysis.
Effects of compound 6b on the phosphorylation
of PI3K/Akt/ERK1/2
in Caco cells. The cells were treated with the 1/4IC50,
1/2IC50, and IC50 for 48 h, and the protein
phosphorylation levels were relative to β-actin protein (internal
control) using western blot analysis.
Conclusions
The condensation of azo pyrazolinone derivatives
with various aromatic
aldehydes yielded a series of novel pyrazolinone chalcones 3–9. This has been confirmed by spectroscopic techniques as well as
elemental analyses. The newly synthesized pyrazolinone chalcone (6b) was selected according to its in silico molecular binding
energy toward PI3K/Akt protein kinases. Consequently, in vitro studies
approved that the chosen compound 6b causes apoptosis
and cell death by inducing ROS generation-mediated inhibition of PI3K/Akt.
The cellular mechanism of interdependence between PI3K/Akt inhibition
and Raf-1/ERK1/2 proliferative inhibition, p53 activation, and cell
cycle arrest involved in the Caco colon cancer cell line has been
elucidated. According to our results, compound 6b could
be used as a promising anticancer agent.
Authors: H M Berman; J Westbrook; Z Feng; G Gilliland; T N Bhat; H Weissig; I N Shindyalov; P E Bourne Journal: Nucleic Acids Res Date: 2000-01-01 Impact factor: 16.971
Authors: Noel M O'Boyle; Michael Banck; Craig A James; Chris Morley; Tim Vandermeersch; Geoffrey R Hutchison Journal: J Cheminform Date: 2011-10-07 Impact factor: 5.514
Authors: Cornelia Braicu; Mihail Buse; Constantin Busuioc; Rares Drula; Diana Gulei; Lajos Raduly; Alexandru Rusu; Alexandru Irimie; Atanas G Atanasov; Ondrej Slaby; Calin Ionescu; Ioana Berindan-Neagoe Journal: Cancers (Basel) Date: 2019-10-22 Impact factor: 6.639
Figure 1
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