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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. |