الفهرس 
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Abstract
Cancer is the second largest cause of death in age below 70
years and it characterized by abnormal cells proliferation. It is a noncommunicable
disease and considered the second most prevalent
cause of death all over the world.
Liver cancer is the most serious and common cancer problem in
Egypt. Hepatocellular carcinoma (HCC) is the predominate type of
primary liver cancer and it is the fourth cause in cancer-related death
in men and sixth in women from malignancy.
Colorectal cancer (CRC) is the third most frequent and
significant reason of cancer mortality in the whole world. Colorectal
cancer developed in individuals who are exposed to a various risk
factors or either have acquired or inherited genetic propensity for the
disease.
Chemotherapy has adverse effects that could damage the tumor
host and the immune system when applied in the systemic way. A
natural product such as herbs, medicinal plants and flavonoids,
alkaloids, and carotenoids forms their phytocompounds. The use of
chemical drugs containing phytocompounds or chemopreventive
substance shows important findings and promising against cancer
fighting. Anti-tumor drugs which made from plant sources, worked in
different ways in order to activate apoptosis in cancer cells.
Cryptolepis Sanguinlenta is a West African plant that has been
used in traditional African medicine, neocryptolepine I and its regioisomer,
cryptolepine are the bioactive indoloquinoline alkaloids
components which extracted from the plant’s roots and has a strong anticancer, antibacterial, antischistosomicidal, and antiplasmodial
effect.
Aim of the work
The aim of the study is to fulfill the cytotoxic activity of the
novel analogue of Neocryptolepine 11(4-Aminophenylamino)
Neocryptolepine, in two cell lines hepatocellular carcinoma (HepG2)
and colon carcinoma (HCT-116), as well, the possible molecular
mechanism through which it exert its cytotoxic activity.
Material and Method
1- Chemistry
First step was the Preparation of the analogue 11-(4-
Aminophenylamino) Neocryptolepine (APAN) compound (salt) in
powder brown state from its parent compound Neocryptolepine and
then structure was affirmed by spectroscopic methods as NMR and
FT-IR, NMR analysis.
2- Modeling studies
Scanning for target was done using Swiss Target moreover,
drug indications were assessed using Soft pred. Smiles were copied
from Chemdraw to the software where calculations were run for target
prediction and indications.
Molecular docking studies were performed using. Two proteins
were downloaded for modeling study from protein data bank
(pdb:6CZ4 and 7JL7) for protein tyrosine kinase 6 and caspase 3
respectively. Ligand and Protein structural optimizations were applied
by calculating partial charges, 3D protonation, strands correction
followed by energy minimization. The selected docking protocol was
induced fit, where the active site was selected at dummy atoms and alpha spheres and backbones were selected as a placement guide.
Exclusion of Pharmacophore annotations was selected. Browsed
database of the investigated compound in mdb format. The gradient
for energy minimization was 0.05, and MMFF94X was the force field
by default.
3- In Vitro
HepG2 and HCT-116 cell lines were obtained, cultured and
treated with 11(4AminoPhenylamino) Neocryptolepine [APAN]
compound after dissolving it in DMSO according to its weight and
some calculations, (control group) cells treated with DMSO, then
IC50 were determined and according to the IC50 cells were treated
with 3 doses depended on IC0, half IC50 and double IC50 the
effective time and dose were determined using MTT assay, on this
base the cells were examined using TEM to get further detection for
APAN activity on both cells, Flowcytometry assay was used to study
the apoptotic activity of APAN on both HepG2 and HCT—116 cell
lines by measuring ANNEXIN V protein (apoptotic marker) and the
last parameter we used was ELIZA to measure the apoptotic proteins
markers Casepas-3 and P53 kits and the Proliferations proteins
markers kit of PCNA, KI-67and VEGF in order to confirm the anticancer,
anti-proliferation activity of APAN in both cell lines.
Results
The scanning showed high affinity for kinases, in particular
protein tyrosine kinase 6 enzyme with range between 60.0 % and
6.7% where kinase represents the highest percentage. With further
investigation to predict the possible indications for APAN using Super
pred software indicated hepatic and colorectal with 92%, which
prompted our interest to apply the antiproliferative studies on HepG2 and HCT-116 cell lines. Molecular docking studies indicated that the
binding affinity scores of APAN for protein PDB code: 6CZ4 of
tyrosine kinase 6 recorded of −6.6084 and RMSD value of 0.8891 °A,
while that for protein PDB: 7JL7 of caspase 3 was -6.1712 and RMSD
of 0.8490 °A. Demonstration of APAN compound in HepG2 and
HCT-16 cell lines induced cytotoxicity by inhibition of cells growth
through reduced cells viability percent in treated cells compared to
control cells which treated with DMSO and existence of cellular
delayed activity and features of early apoptosis elucidated by absence
of cellular membrane, shrunken nucleus, chromatin condensation and
absence cytoplasm organelles comparison to control cells which
displayed a normal state of cancer cells, intact cellular process
containing complete integrity of cells organelle and nuclear membrane
with well-distributed chromatin and nucleus under TEM. As well, a
numerically increase in the protein expression level of Annexin V
(apoptotic protein) compared to control cells, induced Casepas-3 and
P53 the apoptotic marker proteins level in both cells significantly, in
addition a significant reduced in the expression level of proliferation
proteins PCNA, KI-67 in cells and VEGF in the cells medium.