Anti-cancer potency by induced apoptosis by molecular docking P53, caspase, cyclin D1, cytotoxicity analysis and phagocytosis activity of trisindoline 1,3 and 4

Abstract

Cancer is one of the leading causes of death in the world. Efforts to find and develop cancer drugs from natural products continue with the exploration of trisindoline, a substance that is isolated from marine sponges Hyrtios altum. Trisindoline is an indole trimer alkaloid compound that has been successfully synthesized into trisindoline 1, 3 and 4. Trisindoline is cytotoxic in cell lines and in this study, trisindoline was able to induce apoptosis in the in silico and in vitro tests that were carried out. The in silico test was carried out through molecular docking using the Autodock Vina method and the Molecular Dynamics (MD) Simulation QM / MM AMBER. The target proteins used were protein p53 and caspase -9 which played a role in the apoptotic pathway and cyclin D1 which played a role in cell proliferation. Meanwhile, cytotoxicity analysis was carried out using the MTT method (3- (4,5-dimethyltiazol -2-yl) -2,5 -dipenyl tetrazolium bromide). Nevertheless, the ability of trisindoline to induce phagocytosis is still unrevealed. The phagocytosis assay was carried out by assessing the macrophage capacity and phagocytic index using the latex-beads model. The in silico results showed that the binding affinity values between the target protein Cdk-2 and the trisindoline 1, trisindoline 3 and trisindoline 4 ligands were -7.3 kcal / mol, -7.7 kcal / mol and -6.6 kcal / mol respectively. The binding affinity values between the target protein p53 and the trisindoline 1, trisindoline 3 and trisindoline 4 ligands were -7.5 kcal / mol, -7.4 kcal / mol and -7.5 kcal / mol respectively. The binding affinity values between the target protein caspase-9 and the trisindoline 1, trisindoline 3 and trisindoline 4 ligands were -7.5 kcal / mol, -7.1 kcal / mol and -7.2 kcal / mol respectively. The results of RMSD (Root Mean Square Deviation), RMSF (Root Mean Square Fluctuation), and hydrogen bonds in the MD (Molecular Dynamics) Simulation showed that Cdk-2 formed a protein complex with trisindoline 3, protein p53 with trisindoline 1 and caspase-9 with trisindoline 1. The cytotoxicity assay was carried out in the MCF-7 cell line and the IC50 value obtained for trisindoline 1 was 2.059 μM, for trisindoline 3 was 3.9759 ​​μM, for trisindoline 4 was 15.46 μM and for doxorubicin was 9.88 μM. Furthermore, the phagocytosis test was carried out using trisindoline 1, 3 and 4. Our results showed that 6.25 µg mL^-1 of trisindoline 1 and trisindoline 3 were able to induce the phagocytosis capacity of macrophage cells of 38.34; whereas trisindoline 4 at a concentration of 50 µg mL^-1 induces a phagocytosis capacity of 32.89. Trisindoline 1, 3 and 4 showed potentials of immunostimulants at low concentrations but showed potentials of immunosuppressants at high concentrations. The overall results demonstrated that trisindoline 1 and 3 are potential anti-cancer candidates capable of activating the apoptotic pathway.

Keywords: Caspase 9; In Silico; MCF-7; P53; Trisindoline.

Fig. 1

Chemical Structure of Trisindoline (A) Trisindoline 1 (B) Trisindoline 3 and (C) Trisindoline 4.

Fig. 2

(A) Visualization of the molecular docking results of Cdk-2 with Trisindoline 1 (a) and the hydrogen bonds formed (b); (B) Visualization of the molecular docking results of Cdk-2 with Trisindoline 3 (a) and the hydrogen bonds formed (b) and (C) Visualization of the molecular docking results of Cdk-2 with Trisindoline 4 (a) and the hydrogen bonds formed (b).

Fig. 3

(A) Visualization of the molecular docking results of P53 with Trisindoline 1 (a) and the hydrogen bonds formed (b); (B) Visualization of the molecular docking results of P53 with Trisindoline 3 (a) and the hydrogen bonds formed (b) and (C) Visualization of the molecular docking results of P53 with Trisindoline 4 (a) and thehydrogen bonds formed (b).

Fig. 4

(A) Visualization of the molecular docking results of Caspase-9 with Trisindoline 1 (a) and the hydrogen bonds formed (b); (B) Visualization of the molecular docking results of Caspase-9 with Trisindoline 3 (a) and the hydrogen bonds formed (b) and (C) Visualization of the molecular docking results of Caspase-9 with Trisindoline 4 (a) and the hydrogen bonds formed (b).

Fig. 5

(A) RMSD of the Cdk-2 protein complex (B) RMSF of the Cdk-2 protein complex (C) Intermolecular hydrogen bonds of the Cdk-2 protein complex with trisindoline 3.

Fig. 6

(A) RMSD of the Cdk-2 protein complex (B) RMSF of the Cdk-2 protein complex.

Fig. 7

Intermolecular hydrogen bonds of the p53 protein complex with trisindoline 3.

Fig. 8

(A) RMSD Caspase-9 protein complex (B) RMSF Caspase-9 protein complex.

Fig. 9

Intermolecular hydrogen bonds of the Caspase-9 protein complex with trisindoline 3.

Fig. 10

Morphology of macrophage cells in mice (Mus musculus) with a magnification of 400x. A) Normal macrophages, B) macrophages that phagocytes latex particles, C) Latex particles free. Note: n = nucleus, s = cytoplasm, p = pseudopodia, l = latex.

Fig. 11

The morphology of macrophage cells of mice treated with trisindoline with concentrations of (a) 0 μg/mL, (b) 6.25 μg/mL, (c) 12.5 μg/mL, (d) 25 μg/mL, (e) 50 μg/mL, and with doxorubicin (f) with a concentration of 12.5 μg/mL. A magnification of 400x was used. Note: m = macrophages n = nucleus, s = cytoplasm, p = pseudopodia, l = latex.