- Research article
- Open Access
Antitumor effect of proanthocyanidin induced apoptosis in human colorectal cancer (HT-29) cells and its molecular docking studies
© The Author(s) 2019
- Received: 6 July 2018
- Accepted: 16 January 2019
- Published: 4 February 2019
- Cell cycle arrest
- Molecular docking
Colorectal cancer (CRC) is the highest prevailing destructive tumor mortality in worldwide . CRC is treated with surgery, radiation and chemotherapy, depending on the tumor site and disease stage. These chemotherapy drugs are formulated to disrupt tumor cell microtubule signals and hyperproliferative status of these cells has been inhibiting the cell-cycle arrest and subsequently inducing cell death . The cell cycle progression of G1/S and G2/M phases derived from cyclin-dependent kinases (CDKs). The CDKs proteins binding with exact cyclins through sequential activation of cell cycle progression. Cell cycle regulating protein cyclin A is fixed to CDK2 or CDC2 proteins, a crucial aspect in rectifying the S phase transformation.
Hence the several chemotherapeutic phytomedicines, flavonoids have been a comprehensive part in preventing cell generation and transition. Edible vegetables, grains and grass are a fabulous source of flavonoids. These flavonoids have acquired a developing consideration in the field of pharmacology due to their assorted therapeutic effects. The access to phyto-medicine had been adopted, strongly in the therapy for many groups of tumor [3, 4], which produced better chemopreventive effects when they are derived from natural sources. The low concentration of phyto-medicine compound displays successful treatment for treating malignancy cells without influencing typical cells.
Proanthocyanidin (PAC) isolated from the Vitis vinifera, is a well-known phytochemical due to their numerous biomedical application, containing oxidation inhibitor, anti-irritation, neurological, low blood sugar, cardiac disorders, antitumor, etc. [5, 6]. Generally, PAC may prevent a sequence of signal interchange mechanisms identical to cancer causing and behaves as an appropriate receiver of tyrosine enzyme and proteasome . When treated with PAC, a polyphenolic compound, could specifically bind to nucleic acids, and prompt the damage of DNA in tumor cells by controlling the function of DNA topoisomerase and finally affects cell death , that is related among the DNA bruise response (DBR). Hence, DBR causes cell suicide, namely caspase-mediated cell death, whereas DNA loss may not be effectively rectified . The DBR frequently generates autophagic cell destruction in tumor cells. A huge number of studies exposed that (−)-Epigallocatechin gallate (EGCG) plus curcumin shown impressive malignant tumor and therapeutic actions of less toxic following different types of carcinoma over the diverse processes [10, 11]. Hence, this study is aimed to investigate whether the natural compound PAC could exert therapeutic effects, in particular to colon cancer (HT-29) cells.
Computational supported drug screening techniques is frequently used to forecast through an extensive study of ability, this structure binding with the relevant targeted small molecule ligands. Moreover, that can be selected by analyzing the interaction between phytochemicals . The phenomenal research was checked to study the efficiency of the PAC compound to generate more repressive action on HT-29 cells in vitro and in silico analyses.
Proanthocyanidin (PAC) compound was acquired from Sigma-Aldrich (St. Louis, MO, USA). 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT), Propidium iodide (PI), Dulbecco modified eagle’s medium (DMEM), Fetal Bovine Serum (FBS), Penicillin, and Streptomycin, Dimethyl sulfoxide (DMSO), DNA extraction kit was obtained from (Hi-media, Chennai). All the chemicals and reagents utilized were should be more specific.
Human colorectal cancer (HT-29) and normal cell line FHC (human fetal normal colonic mucosa) was procured from National Center for Cell Sciences (NCCS), Pune, India. This cell line was grown in RPMI-1640 (Gibco-BRL) medium containing (10% v/v) warm-suspended fetal bovine serum (Gibco-BRL) additionally 2 mM-glutamine (Sigma Chemical); Penicillin (100 mg/mL); Streptomycin (100 mg/mL). These supplemented media are referred to as complete media or growth media and routinely cultured at 37 °C in a 5% CO2 water-saturated atmosphere. Cells were passaged and subculture to 90% confluence with 0.2% trypsin (w/v) every 2–3 days.
Cell growth inhibitory assay
The survival of treated carcinoma cells was checked through MTT assay. Briefly, cells were patched in 96-well dishes at a viscosity of 4 × 103 cells/mL inside 100 µL supplement and permitted to adhere overnight. After incubation, increasing concentrations (1.56–50 μM) of the PAC was affixed and grown for 24 h. Then 50 µL of MTT (3 mg/mL in PBS) was covered for 4 h at 37 °C. The unbounded solution was detached likewise each wells were added 100 µL of DMSO. The optical density was detected at 570 nm by using small SYNERGY HTX SILFA multi-mode reader (BioTek, USA). The irregular shapes were visualized under bright-field inverted phase contrast light microscope (Nikon, Japan) at 400× magnification.
Acridine orange and ethidium bromide staining
Apoptosis was determined by the detection of nuclear morphology using AO/EtBr staining methodology and it is based on the difference in membrane integrity between apoptosis and necrosis. The sterile coverslip was placed in 6-well tissue culture plates while cells were inoculated at a concentration of 4 × 103 cells/mL. Following incubation, the medium was evacuated and replenished with medium containing FBS (10%) along with the cells processed through PAC compound with 24 h. The cells were disinfected from PBS and rigid with 4% P-formaldehyde for 5 min at 4 °C. Further, cell culture was stained with an AO/EtBr solution (100 µg/mL AO and 100 µg/mL EtBr in PBS) proceeding in dark condition. This suspension was viewed under a fluorescence emitting microscope (Nikon, Eclipse 600, Japan) 400× amplification at 510–590 nm.
The DAPI is a blue fluorescent dye which is sensitive to chromatins and very less toxic to cells, it measured to observe the nuclei changes in apoptotic cells. HT-29 cells were patched in 6-well dishes and maintained at 37 °C along 5% CO2 and incubated for 24 h. The cells were prepared at GI50 absorptions of (3.12 µM, 6.25 µM and 12.5 µM) for 24 h. Then the cells were disinfected using PBS and treated with 4% P-formaldehyde for 15 min, it was then permeabilized with 0.1% Triton X-100 and stained with DAPI (1 mg/mL) for 10 min. The stained cells were pictured using a fluorescence microscope with the suitable excitation filter.
DNA disintegration analysis
DNA disruption research was conducted using an agarose gel electrophoresis method. In brief, appropriate GI50 absorption of PAC activated cells were grown in 10 cm culture plate. Together connected and detached cells were unstained with ice-cold PBS. The cells were thawed in 500 µL of lysis buffer (0.5% Triton X-100, 10 mM EDTA, 10 mM Tris–HCl, pH 8.0 and 2 mg/mL proteinase K) and were treated for 3 h at 55 °C, additionally RNAse A (Amrsco, Solon, OH) was supplemented for 3 h. DNA was extracted with saturated phenol: chloroform (1:1), accelerated using ethanol precipitation along with Tris/EDTA buffer (10 mM Tris–HCl, pH 8.0, and 1 mM EDTA). The detected DNA was visualized in 2% agarose gel electrophoresis, incorporated with ethidium bromide (0.5%), and were observed using UV-trans illuminator system (Bio-Rad).
Analysis of cell cycle progression
To regulate the consequence of PAC and their promoting apoptosis on the cell division in HT-29 cell line, the cells were exposed to the pre-determined GI50 concentrations of test compound were measured with 24 h untreated control cells (considered with drug free media). Cells were harvested and trypsinized, followed by rinsing with PBS and extended in binding buffer. The cells were labelled with propidium iodide in PBS containing RNAse (0.1 mg/mL). The labelled cells were examined under FACS Calibur (Becton–Dickinson, San Jose, CA) flow cytometer. Cell division and apoptotic ratios in Sub-G0, G1/S and G2/M phase were consequently determined using BD Cell Quest Pro Software (Version 5.1).
In silico study
The high performance GPU operated with Cent OS V6.6 were used to study molecular modeling with the help of the Linux operating platform. Computer stipulations of the HPC GPU-Super micro Intel Xeon E5-1620 v3 series of specification 32 GB DDR4-2133 ECC RDIMM of RAM and 2 GB Graphics card of NVIDIA Quadro K620 with 4-core processor. The specific docking software used is a commercial version of the Schrodinger software package, LLC, New York, NY 2015.
For the analysis of docking simulation, the protein complex was retrieved from protein data bank (PDB) as shown in (Table 3). The receptor protein structure for the docking simulation was pre-processed using the Protein Preparation Wizard module in Schrodinger software . The protein residues His, Asp and Glu residue gets into protonated states, when H-atoms are added along with hydrogens on thiols and hydroxyls and were sampled to H-bond optimize network. This Schrodinger provides missing side chain alignment of each residue using the build interface incorporation. The OPLS-2005 force field used to optimize the state subsequently for the minimization process with an Impact Refinement module to condense steric clashes that may exist in the structures. When the average root mean square value of non-hydrogen atoms reached the 0.3 Å, the minimization was terminated .
Proanthocyanidin ligand was prepared for structure optimization and conformer generation using LigPrep . The 2D companion into 3D conformer was converted in all the compounds. The optimized energy conformers of ligand were obtained by the application of OPLS force field. Neutralization of charge groups, addition of implicit hydrogen atoms, tautomerization, generation of various ionization, and chiralities states of the ligand molecule were the prior steps taken before the energy minimization process of ligand structures .
Quantum polarized ligand docking
The partial charges on the ligand atoms was enhanced by quantum-polarized ligand docking (QPLD) in a Glide docking run and replacing them with charges received from quantum automatic prediction. In QPLD using Glide, the chosen ligand was docked and charges on the ligand elicited by the protein are studied, the best ligand interactions are re-docked. Molecular mechanics and quantum mechanics (QM/MM) (efficiency/velocity) combined option was provided by QPLD. The accuracy was enhanced with minimum accomplishing duration with the united QM/MM approach for the atomistic level prediction of binding energy and charge transition. Interaction between ligand and the binding site of protein was done by QM calculations of QPLD and the remaining protein region was calculated by MM force field .
The marginal partial charge cut-off value of 0.25, the vander Waal radii of receptor atoms were scaled with 2.00 Å to soften the potential for non-polar part of the receptor were selected. The charge calculation, selection of QM level is a tradeoff between accuracy and speed. The surface electrostatic potential energy is used for calculating partial charges by accurate and fast modes. Fast uses the B3LYP functional, ‘Quick’ self-consistent field (SCF) and 3–21G basis set of accuracy level. For the functional theory calculation in the QM region accurate uses B3LYP, ‘Ultrafine’ SCF accuracy level (iacc = 1, iacscf = 2) and the 6–31G⁄/LACVP⁄ basis set. Quantum mechanics cover the ligand and active site region and the remaining protein system comes under molecular mechanical mode.
Binding free energy calculation
The GBSA continuum models were carried out for simulations. Gaussian surface is used instead of a van der Waals surface for better representation of solvent-accessible surface area with surface generalized Born (SGB) using by Prime model .
All the statistical calculation was carried through SPSS version 20.00 for Windows. The entire data were communicated as mean ± SE of triplicate independent analysis. One-way ANOVA with Dunnett’s post hoc test for multi-group comparisons (except the GI50 values which were estimated by nonlinear regression analysis using GraphPad Prism software, (Version 5.0). A p-value of 0.05 or less was examined as significant.
Cell growth inhibitory assay
Analysis of cell cycle progression
QPLD analysis of docking scores
Glide XP analysis of proanthocyanidin and 5-fluorouracil
XP analysis of docking scores
Glide E model energy
Glide E model energy
Arg105, Glu95, Asp99
Asp86, Asn132, Glu8
Asp97, Thr177, Lys142
Binding free energy calculation
Binding free energy calculation of protein-proanthocyanidin and 5-FU complex using MM/GBSA method
Binding free energy
∆G Coulomb a
∆G vdw b
∆G solLipo c
∆G bind d
∆G Coulomb a
∆G vdw b
∆G solLipo c
∆G bind d
Proteins chosen for docking with proanthocyanidin
Name of the proteins
In modern approaches in computational system have enabled virtual screening of drug discovery . The amino acid Leu83 and Asp146 in the water-repellent section as a play vital function in regulating with CDK2/cyclin B complex activity . This effect has been approved with the previous statement to evaluate the intensity of CDK2 as a pharmaceutical objective for tumor treatment. The hydrogen bond structure with cyclin B/CDK2 and dockings core indicates that proanthocyanidin has able to suppress the properties of CDK2 protein in cell division are productively than 5-FU. Regarding, the crucial amino acids His95 and Val96 act as the decisive role in CDK4 activation . The post-scoring approach were evaluated by using the docking complexes. MM/GBSA obtain the free energy computation process, which helps a consolidation of molecular logistics intensity and constant resolver modes. In the terms of hydrogen and non-hydrogen bond interactions with protein–ligand complex, such as van der Waals, electrostatic energies, polar and non-polar destruction free forces with the supplement form of entropy . Thus, these proteins were considered as receptors to explore the anticancerous activity of selected polyphenolic compounds. It is observed that the binding affinity of PAC was higher with BCL-XL (− 5.23 kcal/mol) and CDK2 (− 5.17 kcal/mol), when compared to the other proteins of BCL2 and CDK4. Interestingly, these results are in agreement with the in vitro and in vivo analyses, which indicated that PAC is having a better inhibition activity of these compounds is found to be higher when compared to 5-FU. In our experience, this is the first report to explore the anti-cancer activity of PAC against Colon cancer.
The present study displayed the therapeutic activity of PAC as an effective anticancer agent on colon cancer (HT-29) cells. The cell growth and AO/EtBr staining exposed the linguistic difference of PAC compound promotes the apoptotic cells with chromatin condensation and membrane blebbing in HT-29 cells. The cell cycle arrest G2/M phases and percentage of apoptotic cells was confirmed by the dose-dependent manner and apoptotic cell death. Hence we suggest that this flavonoid compound can be considered as an effective anticancerous promoter against human colon cancer cells.
MS, PP and BMG designed and performed the research. BR, AS, PP analyzed the data and interpreted the results. MS wrote the paper. All authors read and approved the final manuscript.
The authors appreciatively concede the sponsored program of DST–INSPIRE, Department of Science and Technology, New Delhi (REF. NO: DST/INSPIRE Fellowship/2015/IF150459). The authors also acknowledge the Principal, CEO and the Head of Department, Department of Biotechnology, K. S. Rangasamy College of Technology for their support offered towards the study. We also thank the DST–FIST (fund for infrastructure for science and technology) FIST NO: 368 for their support in providing the facilities, DBT–STAR scheme for their basic support on instrumentation to the Department. The authors would like to extend their sincere appreciation to the Deanship of Scientific Research at King Saud University for funding this Research Group No. (RG-1437-024). Also the author thanks Sejong University, and Kyonggi university, South Korea for the valuable contribution.
The authors declare that they have no competing interests.
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