Antitumor effect of proanthocyanidin induced apoptosis in human colorectal cancer (HT-29) cells and its molecular docking studies

Abstract

Proanthocyanidin (PAC) is a promising compound that has displayed its potent antineoplastic properties with a specific intrinsic pathway. This precise us to explore the phyto-preventive effect of PAC against colon cancer (HT-29). The results showed that PAC inhibited the cell growth and GI₅₀ value was found to be 6.25 μM for 24 h exposure, when correlated to the normal cell line does not have toxicity was noticed. The linguistic differences, similarly membrane blebbing, cell shrinkage fragmented nuclear bodies and mitochondrial membrane were observed in AO/EtBr and DAPI staining. The features of regular mechanical apoptotic characterization was analyzed by DNA fragmentation. The cell cycle arrest at G2/M phases was detected using FACS analysis. The early and late apoptotic cells were observed by using Annexin V/PI staining. The ligand-protein interaction and docking studies were performed using Schrodinger's software. The QPLD analysis of docking studies revealed that PAC exhibited better binding affinity of - 5.23, - 5.17 and - 4.43, - 4.47 kcal/mol against BCL-XL, CDK2 and were compared with 5-FU respectively, which significantly reveals the anticancerous activity of Proanthocyanidin compound. Thus, the PAC compound provides future application of therapeutic option in the treatment of colon cancers.

Keywords: AO/EtBr; Cell cycle arrest; Molecular docking; Proanthocyanidin.

Fig. 1

Cytotoxic effect of PAC exerts against Colon cancer (HT-29) cells. Cells were treated with indicated concentrations of PAC alone for 24 h. Cell viability was detected by MTT assay. The experiments were carried out in triplicate and each value represents mean ± SE

Fig. 2

Light microscopic image of HT-29 cells treated with PAC for 24 h. Affected cells showed some morphological characteristic of apoptosis such as cell shrinkage and membrane blebbing at (×400) magnification

Fig. 3

Nuclei morphological changes seen in HT-29 cells stained with AO/EtBr fluorescence microscopy. HT-29 cells were seeded in 6-well plates (4 × 10³ cells/mL) and treated with PAC compound with GI₅₀ concentration respectively for 24 h at 37 °C

Fig. 4

Analysis of DNA fragmentation induced by PAC in HT-29 cells for 24 h. Control cell lines incubated with DMEM medium alone. DNA fragmentation was assessed by 1.5% agarose gel electrophoresis and ethidium bromide staining and viewed under a UV transilluminator. Fragmented internucleosomal DNA appears as a ladder. “M” represent 1 bp DNA marker, (L1) Untreated, (L2) 3.12 µM, (L3) 6.25 µM and (L4) 12.5 µM

Fig. 5

Cell cycle analysis of PAC induced apoptosis in HT29 cells by using Flow Cytometry. a Untreated cellular DNA was stained with PI and the distribution of the cells in G0/G1, S, and G2/M phase analyzed, b 3.12 µM, c 6.25 µM and d 12.5 µM

Fig. 6

Treatment of PAC compound induces apoptosis in HT-29 cells were analyzed using Annexin V-FITC/PI double staining assay

Fig. 7

Glide XP docking interaction poses of proanthocyanidin (PAC) and 5-FU with BCL₂ a PAC, b QPLD docking poses of PAC, c 5-FU and d QPLD docking poses of 5-FU

Fig. 8

Glide XP docking interaction poses of proanthocyanidin (PAC) and 5-FU with BCL-XL a PAC, b QPLD docking poses of PAC, c 5-FU and d QPLD docking poses of 5-FU

Fig. 9

Glide XP docking interaction poses of Proanthocyanidin (PAC) and 5-FU with CDK2 a PAC, b QPLD docking poses of PAC, c 5-FU and d QPLD docking poses of 5-FU

Fig. 10

Glide XP docking interaction poses of proanthocyanidin (PAC) and 5-FU with CDK4 a PAC, b QPLD docking poses of PAC, c 5-FU and d QPLD docking poses of 5-FU