Apoptosis induction of kadsuric acid from Vietnamese Kadsura coccinea (Lem.) A. C. Smith in human pancreatic cancer cells: in vitro and in silico approach

Apoptosis induction of kadsuric acid from Vietnamese Kadsura coccinea (Lem.) A. C. Smith in human pancreatic cancer cells: in vitro and in silico approach | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Apoptosis induction of kadsuric acid from Vietnamese Kadsura coccinea (Lem.) A. C. Smith in human pancreatic cancer cells: in vitro and in silico approach Tan Khanh Nguyen, Manh Hung Tran, Tan Trung Truong, Long-Hung Dinh Pham, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4328289/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 11 Sep, 2024 Read the published version in Revista Brasileira de Farmacognosia → Version 1 posted 5 You are reading this latest preprint version Abstract Kadsuric acid, a major triterpenoid isolated from the leaves of Vietnamese Kadsura coccinea , exhibited potent cytotoxic effects in some human cancer cells. In this study, the effects of kadsuric acid on pancreatic cancer cells PANC-1 were investigated. The results showed that kadsuric acid exhibited dose-dependent cytotoxicity against PANC-1 with an IC50 value of 14.5 ± 0.8 µM. Kadsuric acid effectively activated caspase-3 by increasing the level of enzyme cleavage by 1–2 times after 12 and 24 h, and by more than 3–4 times compared to the negative control. In addition, this compound enhanced both two types of cysteine-aspartic acid proteases, including caspase-3 and caspase-9 through protein expressions. Western blot analysis also indicated that kadsuric acid reduced Poly [ADP-ribose] polymerase 1 (PARP1) expression in PANC-1 cells. For underlying mechanism insights, molecular modeling methods were applied to investigate the binding interaction between kadsuric acid and PARP1. Compared to the co-crystallized ligand, kadsuric acid displayed a stronger binding affinity (-9.3 kcal/mol). A molecular dynamics simulation showed that the complex is stable over 200 ns. Taken together, it can be determined that kadsuric acid can interact with the DNA of human pancreatic cancer cells through the intrinsic caspase/PARP-1 pathway. This study can guide future research on kadsuric acid as PARP1 inhibitor for cancer treatment. Kadsura coccinea kadsuric acid caspases PARP1 pancreatic cancer molecular modeling Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction The advancement of computational biology has provided new opportunities for research in the development of medicines for the health sciences. In particular, in silico techniques have become increasingly important in exploring the biological effects of potential active ingredients on the human body(Roy et al., 2017 ). In silico methods can be used to identify drug targets, evaluate interactions between compounds and proteins, perform pharmacokinetic analysis (ADME), and improve the drug properties of molecules (Nguyen, Nguyen, et al., 2022 ; Nguyen, Tran, et al., 2022 ). These techniques have already proven to be highly effective in research and are expected to pave the way for new drug development projects (Nguyen, Tran, et al., 2022 ). Poly ADP-ribose Polymerase 1 (PARP1) is an enzyme found in the nucleus that is important for regulating various cellular processes, including DNA repair, transcription, and chromatin remodeling (Dadheech et al., 2022 ; Tao & Wu, 2021 ). It belongs to a family of enzymes called ADP-ribosyltransferases, which modify proteins by adding ADP-ribose units. In cancer treatment, inhibiting PARP1 prevents cancer cells from repairing DNA damage, ultimately leading to cell death. However, normal cells with functional DNA repair mechanisms can tolerate PARP1 inhibition (Li et al., 2020 ). Several PARP1 inhibitors, including olaparib, rucaparib, niraparib, and talazoparib, have been approved for the treatment of various types of cancer, particularly ovarian and breast cancer, and have shown promising results in clinical trials (Li et al., 2020 ; Tao & Wu, 2021 ; Zhang et al., 2022 ). Kadsura coccinea (Schisandraceae) is a climbing vine with a woody, brown-black stem, and smooth branches. It is found in high mountain areas with tropical or subtropical climates in India, Laos, Vietnam, and southern China (Pham Hoang Ho, 2006 ). Folk experience shows that K. coccinea is used as a tonic, activating blood, alleviating pain, and stimulating digestion. The fruits are edible when ripe, the seeds are sometimes used as a replacement for the medicine Schisandra chinensis (Y. Yang et al., 2020 ). According to previous studies, the main chemical components of K. coccinea are lignan and triterpenoid compounds. Many lignans have been found in this plant with dibenzocyclooctadiene structures such as kadsutherin, isokadsuranin, kadsurins (Long et al., 2022 ; Woo et al., 2020 ; Y. Yang et al., 2021 ; Q. J. Zhao et al., 2014 ; T. Zhao et al., 2021 ), among them, some substances could inhibit the formation of nitric oxide (NO) during the inflammatory process and anti-cancer (Daniyal et al., 2021 ; Lu et al., 2022 ; Y. Yang et al., 2021 , 2022 ; Q. J. Zhao et al., 2014 ; T. Zhao et al., 2021 ). The triterpenoids were isolated from this plant (Hu et al., 2015 , 2016 ; Liang et al., 2013 ; Xu et al., 2019 ; Y. Yang et al., 2020 ), and were showed to inhibit the growth of human leukemia cancer cells (Daniyal et al., 2021 ; Tram et al., 2022 ; Wang et al., 2012 ). In addition, extracts of K. coccinea exhibited hepatoprotective effects in mice, anti-gastric cancer, and other several pharmaceutical effects (Daniyal et al., 2021 ; Liu et al., 2014 ; Y. P. Yang et al., 2021 a). However, in Vietnam, there are very few scientific studies on the chemical composition and biological effects of K. coccinea (Ban et al., 2009 ). Our previous research showed that K. coccinea is a valuable plant source capable of inhibiting the growth of many carcinoma cell lines, including two pancreatic cancer cell lines PANC-1 and MIA PACA. In addition, we isolated and determined the chemical structure of several lanostane triterpenoids from the leaves of K. coccinea including kadsuric acid as a main phytochemical component (Le et al., 2018). In this study, we reported the cytotoxic effects of this natural compound on PANC-1 cancer cells via the integration of in vitro and in silico methods. 2. Materials and methods 2.1. Kadsuric acid sample Kadsuric acid: White amorphous powder; [α] 24 D + 68º (c 0.25, MeOH); EI-MS m/z 470.3 [M] + C 30 H 46 O 4 . 1 H-NMR (CDCl 3 , 400 MHz) and 13 C-NMR (CDCl 3 , 100 MHz) were described in detail in our previous publication (Le et al., 2018). The chemical structure of kadsuric acid is shown in Fig. 1 a. 2.2. Equipment and instruments used in cell toxicity experiments Equipment and instruments used in cell toxicity experiments included 96-well plates (SPL Life Sciences, Korea), centrifuge tubes 1.5 mL, 2.0 mL, surface treated culture plate, multichannel pipette (Isolab, Germany), hemocytometer, cell counting chamber (improved Neubauer); Panasonic CO 2 incubator (MCO19AIC), ELISA reader machine (ELISA Bio-Rad machine, USA), autoclave sterilization, and drying cabinet. Chemicals used in evaluating the cytotoxic effects on cancer cells include Dulbecco's modified Minimum Essential Medium (DMEM), Roswell Park Memorial Institute (RPMI) 1640 Medium (Gibco, USA), phosphate buffer saline (PBS) (Gibco, USA), fetal bovine serum (FBS) (Gibco, USA); MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (DUCHEFA biochemie, Netherlands). 2.3. Cancer cells and cell cultures The human pancreatic cancer PANC-1 cell lines were provided by Prof. Jeong-Hyung Lee, Kangwon National University, Korea. The cell lines were cultured at 37°C in DMEM supplemented with 10% Fetal Bovine Serum (FBS), 100 U/mL penicillin, and 100 mcg/mL streptomycin in a 5% CO 2 incubator for 48 h. 2.4. Evaluate caspase 3 activation Caspase-3 is an enzyme in the caspase family (cysteine-aspartic protease enzymes) that normally exists as an inactive precursor in cells. When this enzyme is activated, it initiates the process of cell death (apoptosis) by cleaving a protein (substrate) that specifically targets the amino chain DEVD (Asp-Glu-Val-Asp). Ac-DEVD-AFC is a substrate labeled with a fluorescent molecule, that is cleaved by caspase-3 at the bond between D and AFC (7-amino-4-trifluoromethylcoumarin) as entering a cell, thereby releasing the fluorescent AFC that can be quantified by fluorescence spectrophotometer (Tuan et al., 2019 ; Vo et al., 2021 ). Cancer cells were treated with reagents at various concentrations from 1 to 30 µM. After incubation for 12, 24, and 48 h, the cells were harvested, washed with cold PBS, and then lysed in a lysis buffer at room temperature for 10 min, then cooled and added to a test buffer in each well, incubated at 37°C for 1 h. The fluorescence intensity was measured by stimulation at 370 nm and emission at 505 nm using a Twinkle LB970 machine (Berthold Technologies, Germany). The sample containing only cancer cells without compound perturbation was used as the control group. The extent of caspase-3 enzyme induction was determined by the ratio of the fluorescence intensity of the test sample to that of the control sample. The results were processed using GraphPad Prism 3.03 software. 2.5. Molecular docking The crystal structures of PARP1 (PDB ID: 5WS1) were downloaded from Protein Data Bank (RCSB PDB). The preparation process was following our previous study (Tran et al., 2022 ). The process of removing unrelated co-crystallized ligands and water molecules was done by using the Discovery Studio 2020 Client software. After that, the protein structure was minimized using SwissPDB. Then, molecular hydrogens and Kollman charges were added to the protein by the AutoDock Tool software version 1.5.6 (Morris et al., 2009 ). The active site of protein was also selected according to the interactive site of co-crystallized ligand and protein. The grid box site was set at 18.75 × 18.75 × 18.75 for x,y and dimensions. For ligand preparation, the 3D structures of the compound were downloaded from the academic structural library PubChem. Then, Open Babel software was used to convert to .pdbqt format for docking process. The application Autodock Vina version 1.2.3 was implemented to carry out the molecular docking of PARP1 (Eberhardt et al., 2021 ). The docking score was presented in kcal/mol. 2.6. Density Functional Theory (DFT) The DFT/B3LYP method (Becke, 1993 ) with a 6-311G(d,p) basis set in the gas phase was used in these calculations. The highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) energy have been computed. Furthermore, the energy gap (Eg) and several other important parameters, which as ionization potential ( IP ), electron affinity ( EA ), electronegativity (χ), chemical hardnesses (η), electronic chemical potential (µ), chemical softness (S), and electrophilicity (ω) were calculated by using Eqs. (1)-(8) (Medoro et al., 2023 ). Eg = E LUMO - E HOMO (1) IP = - E HOMO (2) EA = - E LUMO (3) χ = -(E LUMO + E HOMO )/2 (4) η = (E LUMO - E HOMO )/2 (5) µ = -χ = (E LUMO + E HOMO )/2 (6) S = 1/2η (7) ω = µ 2 /2η (8) The molecular’s electrophilic/nucleophilic reactive location was determined using molecular electrostatic potential (MEP). All calculation was carried out with the Gaussian 16 program (Frisch, 2016 ) and data from Gaussian output files were visualized with the help of the program GaussView 6.0 (Dennington, 2016 ). 2.7. Molecular dynamic simulation The protocol of simulation was performed following our previous study (Bich et al., 2023 ). Molecular dynamics simulations were performed using GROMACS 2020.4 in order to further investigate the stability of the ligand in complex with PARP1 (Abraham et al., 2015 ; Pronk et al., 2013 ). The complex’s topology was prepared under the CHARMM36 forcefield and solvated in a truncated octahedral box containing TIP3P water molecules using the CHARMM-GUI server (Jo et al., 2008 ). To ensure a fully stable system, the objective values of 300 K temperature and 1 bar pressure were reached. Finally, the molecular dynamics simulation was run for 200 ns for the complex. Post-simulation principal component analysis (PCA) was implemented using in-built GROMACS modules gmx covar , gmx anaeig , and gmx sham . First, the covariance matrix of protein atoms' trajectories was calculated and diagonalized using gmx covar . Trajectories were then projected on the two largest eigenvectors (named PC1 and PC2) for visualization and further predicted of Gibbs free energy landscape using gmx sham . 2.8. Calculate Binding Free Energy (MM/PBSA Calculations) Molecular mechanics Poisson–Boltzmann surface area (MM/PBSA) has been regarded as a competitive method to estimate the relative binding free energies for the compound and to evaluate the relative stability of the apoprotein. In this investigation, the MOLAICAL script was used to implement the MM/PBSA model to measure the relative binding free energy (Bai et al., 2021 ). 2.9. Western Blot assay Protein-to-protein hybridization (antigen to antibody), antigenic protein is detected by coloration or fluorescence reaction. In Western Blot, a protein mixture was separated by SDS-PAGE electrophoresis. Protein lines were transferred onto the hybrid membrane and individual protein bands were detected by immersing the membrane with primary and secondary antibodies labeled with enzymes or radiolabeled specific for the protein of interest. If the protein of interest is bound by a radiolabeled antibody, its position on the spots can be detected by placing the film on an X-ray film. In our experiment, the cells were incubated with the inhibitor for 24 h at 37°C. Cell fragments were immersed in a lysis buffer containing a mixture of protease inhibitors, and centrifuged to remove the precipitate. Measure the concentration of the supernatant. This protein supernatant was separated by polyacrylamide electrophoresis with SDS (SDS-PAGE) and then transferred to a PVDF membrane filter (Bio-Rad, USA). Place the membrane in 5% nonfat milk powder in Tris buffer containing 0.1% Tween-20 (TBS-T) at 4°C overnight and incubate with the primary antibody at room temperature. Membranes were washed three times with TBS-T and permeabilized with HRP-labeled secondary antibodies (Horse-radish peroxidase), incubated at room temperature for 1.5 h, and then washed three times with TBS-T. Detection of immunological proteins and X-rays. Protein was normalized by repulsing the same membrane with the detection of anti-β-actin antibodies, the membranes were immersed in a rejection buffer at room temperature for 20 min (Vo et al., 2021 ). 3. Results and Discussion 3.1. Activation of enzyme caspase 3 and western blotting analysis The chemical structure of Kadsuric acid is shown in Fig. 1 a. This compound is a 3,4 seco-lanostane triterpene and is present in large amounts from Kadsura coccinea species (Le et al., 2018; Liang et al., 2013 ; Y. Yang et al., 2020 ; Y. P. Yang et al., 2021 b). Kadsuric acid exhibited the cytotoxic activity against PANC-1 with an IC 50 value of 14.5 ± 0.8 µM. To investigate the cytotoxicity mechanism, this substance was consequently selected for testing on caspase-3 activation and Western Blot analysis on PANC-1 cells. PANC-1 cells were incubated with kadsuric acid (1–30 µM) at 37°C with caspase-3 substrate (Ac-DEVD-AFC) for 1h. The fluorescence intensity of the cell lysates was measured to determine caspase-3 activity. The negative control sample contained DMSO 0.1%. Data are presented as the mean ± SD of the results from three independent experiments (*p < 0.01; **p < 0.05). Then, cells were treated with kadsuric acid (1–30 µM) for 48 h. Protein 50 µg/lane from cell lysates were electrophoresed on SDS-PAGE gels and then transferred to total blot PVDF membranes. β-actin was used as an internal control, (–), 0.1% DMSO-treated cells. Activation of caspase-3 leads to the activation of other caspases, such as caspase-6 and caspase-7, which cleave multiple protein structures and regulate protein conformational transformation, which is crucial for cell survival and maintenance. Among caspase family enzymes, caspase-3 was identified as the most important enzyme (executioner enzyme) and is activated by both endogenous and exogenous pathways in the cell (Brentnall et al., 2013 ). Therefore, activating the caspase-3 enzyme will lead to cell death, which is the main mechanism of current cancer treatments. The results showed that kadsuric acid activated caspase-3, increasing the level of enzyme cleavage by 1–2 times after 12 and 24 h, and increasing the cleavage, the activation of caspase-3 by more than 3–4 times compared to the negative control PANC-1 cells that were not cleaved (p < 0.05, Fig. 1 b). These results suggest that kadsuric acid can increase the activation of caspase-3 in the PANC-1 cell line, depending on the concentration and incubation time in the cell culture medium. In next process, kadsuric acid was validated for the direct interaction with PAPR1, which is the target protein in caspase signaling. We also used cleaved poly (ADP-ribose) polymerase (cleaved poly (ADP-ribose) polymerase, PARP) treated with kadsuric acid (1–30 µM) and the protein expression was expressed by western blot analysis. As shown in Fig. 1 c, kadsuric acid induced transformation of PARP inducing the cleaved form of PARP (cleaved PARP) on PANC-1 cells after 48 h. PARP is a protein that plays an important role in helping damaged cells repair themselves. For normal cells, PARP has a role in self-repairing and repairing damage when DNA is broken during the repair process. However, for cancer cells, PARP has a role in the self-repairing of damaged DNA in cancer cells when exposed to foreign agents, which allows cancer cells to be resistant to agents such as drugs or chemotherapy and continue to divide (Alemasova & Lavrik, 2019 ; Li et al., 2020 ). Therefore, for cancer cell lines, drugs that inhibit the activity of PARP protein can slow or stop tumor growth. In this experiment, kadsuric acid played an inhibitory role and changed the structure of PARP into a cleaved protein, whereby PARP would not be able to perform its repair function to restore the damaged DNA of cancer cells. This is the main mechanism of PARP pathway anticancer drugs. Analysis of the cleaved PARP protein content showed that kadsuric acid (1–30 µM) modifies the PARP protein composition and interferes with PARP's ability to repair itself, inducing PANC-1 cell apoptosis in a dependent manner of concentration. To confirm the downstream activity of PAPR1 inhibitor, the effect of kadsuric acid (1–30 µM) on caspase-3 and caspase-9 activation was further examined in the PANC-1 cancer cell line (Fig. 1 d). Typically, caspase-3 exist as inactive precursors of 32 kDa (procaspase-3). When activated, caspase-3 converts to an activated form (activated caspase-3) and causes the cleavage of many DNA fragments in cancer cells, which can induce cell death by initiating the apoptosis pathway through the cleavage of DNA and proteins into distinct apoptotic fragments. While caspase-3 is considered the executioner of cell death, caspase-9 also participates in the apoptosis process of cells, this enzyme is encoded by the CASP9 gene, which is the initiator of cancer cell death. Our result showed that kadsuric acid (1–30 µM) activated procaspase-3 and procaspase-9 to their active forms. Accordingly, when increasing the concentration of these fractions, the expression of caspase-3 and caspase-9 enzyme proteins also increased. This could lead to the conclusion that both segments induce apoptosis of PANC-1 cells. From these findings, we demonstrated that kadsuric acid induced apotosis through activating capase-3 pathway by interacting directly PARP1. 3.2. Molecular docking analysis To further investigate the interactions between kadsuric acid and PARP1, molecular docking was applied. PARP-1 enzyme binds to DNA via transfer chains of ADP-ribosyl moieties (PARs) and two zinc finger motifs from nicotinamide-adenine-dinucleotide (NAD+) to chromatin-associated acceptor proteins. This enzyme plays an important role in promoting DNA repair in cancer cells (Houtgraaf et al., 2006 ). Therefore, the search for compounds with inhibitory activity of PARP1 is considered an effective approach for the development of cancer drugs. In this study, kadsuric acid was docked at the catalytic site of PARP1 to evaluate the binding ability and predict the anticancer mechanism of this compound. The 3D structure of domain of PARP1 (PDB ID: 5WS1) was selected for screening with 352 amino acids from His660 to Thr101. The grid box was set to cover the active site of PARP1 following the interactive site of co-crystalized ligand. To confirm the reliability of the chosen protein structure and docking procedure, the 2-[(3R)-3-azanylpyrrolidin-1-yl] carbonyl-1H-benzimidazole-4-carboxamide co-crystal ligand was subjected to re-docking. This involved analyzing the root mean square deviation (RMSD) between the docking pose and the crystal pose. The findings showed that the docking position had comparable conformation and orientation to the crystal pose, with an RMSD value of 0.525 Å (Fig. 2 a). The results revealed kadsuric acid had docking score of – 9.3 kcal/mol, which was better than the docking score of the reference compound, − 8.6 kcal/mol. The results are presented in Table 1 . The crystal ligand showed interaction with PARP1 with two hydrogen bonds at His862, Ser904 residues and three hydrophobic interactions at Tyr907, Ala898, and Gly863 residues. Meanwhile, kadsuric acid was found to interact with enzyme with three hydrogen bonds at Ser864, Asp770, and Glu763 residues and six hydrophobic interactions at His862, Leu877, Ile872, Tyr896, Tyr907, and Met890 ( Fig. 2 b). In particular, acid residues of the triad, His862, and Tyr896, are required for binding of NAD+, while Glu988, besides substrate positioning, is also critical for catalysis. Notably, similar to the reference compound, kadsuric acid showed interactions with key amino acid residues of the donor site His862, and Tyr896 that play an important role in PARP1 activity (Alemasova & Lavrik, 2019 ). Table 1 Docking results of candidates towards PARP1 protein Compound Docking score (kcal/mol) Hydrogen bond Hydrophobic interaction Kadsuric acid − 9.3 Ser864, Asp770, Glu763 His862, Leu877, Ile872, Tyr896, Tyr907, Met890 2-[(3R)-3-azanylpyrrolidin-1-yl] carbonyl-1H-benzimidazole-4-carboxamide a) − 8.6 His862, Ser904 Tyr907, Ala898, Gly863 a) Reference compound. 3.3. Density Functional Theory (DFT) The frontier molecular orbitals (HOMO and LUMO) of the optimized structure (Kadsuric acid) are presented in Fig. 3 a. Calculated geometric parameters ( Table S1 ) of Kadsuric acid compound are given in Supplementary . Table 2 shows detailed quantum chemical parameters of Kadsuric acid compound by DFT/B3LYP/6-311G(d,p) method. The computed energy value of HOMO (E HOMO ) is -6.370 eV and the energy of LUMO (E LUMO ) is -0.128 eV. The energy gap (Eg) between HOMO and LUMO of the Kadsuric acid molecule is 6.242 eV, lying on the transition of an insulator (> 9 eV) and a semiconductor (< 3.2 eV). This energy level can be interpreted as favorable to intermolecular binding to protein structures (Van Chen et al., 2022 ). Additionally, the reactivity descriptors properties such as electronegativity (χ), chemical hardnesses (η), electronic chemical potential (µ), chemical softness (S), and electrophilicity (ω) were also calculated. Furthermore, molecular electrostatic potential (MEP) was also calculated at B3LYP/6-311G(d,p) level of theory to forecast the electrophilic/nucleophilic reactive sites of Kadsuric acid. In this, the region having the negative potentiality (deep red) over oxygen atoms (O2 and O4) and positive potentiality (deep blue) over hydrogen atoms (H76 and H80) which suggests for the electrophilic and nucleophile attack the respected region, respectively, as shown in Fig. 3 b. Altogether, the findings from quantum chemistry investigations suppose Kadsuric acid has good interactions with protein. Table 2 The calculated quantum chemical parameters of the investigated compound by using DFT Parameters Kadsuric acid B3LYP/6-311G(d,p) (eV) Energy of HOMO (E HOMO ) -6.370 Energy of LUMO (E LUMO ) -0.128 Energy gap (E g ) 6.242 Reactivity descriptors Ionization potential (IP) 6.370 Electron affinity (EA) 0.128 Electronegativity (χ) 3.249 Chemical hardnesses (η) 3.121 Electronic chemical potential (µ) -3.249 Chemical softness (S) 0.160 Electrophilicity (ω) 1.692 3.4. Molecular dynamics simulation Small molecules interacting with protein surfaces can induce large tertiary structure changes, which can be exploited in drug design improvements. The main advantage of molecular dynamics simulation is its ability to identify the stability and flexibility of the protein-ligand complex. This method was used to precisely evaluate the thermodynamics and kinetics of drug-enzyme binding. The docking pose with the highest docking score was selected for the simulation process. The 200 ns trajectories of the complex between kadsuric acid and PARP1 were analyzed to evaluate the stability of the ligands and protein. The findings demonstrated that the structure maintained its stability over the course of the simulation with fluctuation within the range of 1 Å (Fig. 4 a). The RMSF results also showed that the ligand stabilized a number of interaction-related residues at the active site (Fig. 4 b). The distribution of a protein’s atoms along its axis is referred to as the radius of gyration (Rg) (Fig. 4 c). The solvent-accessible surface area (SASA) facilitates the visualization of the protein’s conformational change over ligand binding. Both values fluctuate lightly with values of 0.8 Å (for Rg) (Fig. 4 c) and 200 Å 2 (for SASA) (Fig. 4 e), thus confirming the stability of the protein and the complex. It is highlighted that the total number of hydrogen bonds formed between ligand and protein during 200 ns was also calculated. During the simulation process, the ligand almost always formed 2 to 3 hydrogen bonds with the protein (Fig. 4 d). The average center-of-mass distance between the ligand and the protein during simulation time is shown in (Fig. 4 f). The result indicated the ligands showed a stable distance with less than 1.5 Å of fluctuation. Trajectory PCA analysis revealed a relatively smooth and well-clustered transition of states during 200 ns (Fig. 5 ). The Gibbs free energy landscape also ranged moderately from 0–9.43 kJ/mol, with a majority in the low-energy zone (colored green), thus once again suggesting favourable conditions. 3.5. Binding free energy At the final stage of simulation, the binding free energy of kadsuric acid further explained the potency of kadsuric acid. The different energy components contributing to binding energy are shown in Table 3 . These results confirmed the molecular docking results and demonstrated that kadsuric acid can maintain strong binding with PARP1. Table 3 Components of binding free energy calculation Compound Van der Waal energy (kJ/mol) Electrostatic energy (kJ/mol) Polar solvation energy (kJ/mol) SASA energy (kJ/mol) Binding energy (kJ/mol) Kadsuric acid -166.690 ± 16.894 -161.417 ± 32.083 218.225 ± 22.584 -22.607 ± 1.502 -132.489 ± 28.539 4. Discussion Apoptosis is a form of cell death (programmed death) that occurs under pathological conditions in organisms and contributes to cell replacement, tissue regeneration, and elimination of damaged cells under adverse normal conditions (Brentnall et al., 2013 ; Chaudhry et al., 2022 ). Most cancers are caused by abnormal changes in the genetic material of cells during development. Abnormalities may be caused by the effects of carcinogenic chemicals such as cigarette smoke, radiation, chemicals, or infectious agents. Genetically, cancer is a random error in DNA replication or is inherited, so it can be present in all cells from birth (Houtgraaf et al., 2006 ; Yam et al., 2022 ). In fact, a complex interaction between carcinogens and the host genome may account for cancer development immediately after cell exposure to a stimulus. There are many types of activation to initiate apoptosis, including activation of the caspase enzyme system (Brentnall et al., 2013 ; Yosefzon et al., 2018 ). Scientists have found that one of the key mechanisms is that caspase enzymes are activated prior to mitochondrial changes (Brentnall et al., 2013 ). Studies have shown that activation of the enzyme caspase-9 followed by activation of caspase-3 initiates apoptosis. Caspase enzymes are also able to cleave many other structural forms, including cytoskeletal, nuclear, and proteomic factors, lamin, actin, and conjugated ADP-ribose (PARP) (Brentnall et al., 2013 ). The cleavage of these proteins is associated with cellular morphological alterations that have been demonstrated extensively during apoptosis (Chaudhry et al., 2022 ). There were many studies on the application of natural medicinal herbs and active ingredients from plants to destroy cancer cells and support the treatment of cancers (Cragg et al., 2005 )(Kinghorn et al., 2009 ). Some of the commonly used cancer drugs in the podophyllins group are etoposide and teniposide. The two main components of Catharanthus roseus , vinblastine, and vincristine, have been shown to have the ability to kill cancer cells. Another important compound is paclitaxel (Taxol) (Cragg et al., 2005 ; Gordaliza, 2007 ). Taxol found in the bark of the Taxus brevifolia was used in the treatment of ovarian, breast, lung, pancreatic cancers (Cragg et al., 2005 ; Kinghorn et al., 2009 ). In our experiment, the effect of kadsuric acid on the activation of caspase 3 showed that PANC-1 cancer cells may have been killed within 48 h when treated with kadsuric acid. Normally, caspase 3 exists as an inactive precursor with a size of 32 kDa (procaspase 3) (Brentnall et al., 2013 ). Caspase 3 was converted to an activated form and caused the cleavage of multiple segments of cancer cell DNA, which can induce cell death by initiating apoptosis through the cleavage of DNA proteins into heterozygous compounds. While caspase 3 is considered a cell killer, caspase 9, another cysteine-aspartic acid protease, is also involved in cell apoptosis by encoding the CASP9 gene, which is the initiator of the death cells (Brentnall et al., 2013 ; Yosefzon et al., 2018 ). In this study, we discovered that kadsuric acid from K. coccinea also inhibited the growth and development of PANC-1 pancreatic cancer cells. Kadsuric acid is a substance with the structure 3,4 seco-lanostane triterpene, this secondary metabolite showed to interact with PARP1, PARP1 was then cleaved by caspase 3 into an N-terminal 25 kDa and a C-terminal 85 kDa. The PARPs are a family involved in catalyzing the transport of ADP-ribose to target proteins. PARPs play important roles in various cellular processes, including modulation of chromatin structure, transcription, replication, recombination, and DNA repair. The role of PARP proteins in DNA repair is of particular interest, with the task of detecting errors in the recombination mechanism, PARP repairs cellular DNA to maintain the ability to recombine and divide. Most of the PARP inhibitors used in cancer therapy follow this mechanism. Our study showed that kadsuric acid showed the ability to activate the caspase enzyme system consisting of 2 types of cysteine-aspartic acid proteases, caspase 3 and caspase 9. Through western blot protein expression, these enzymes were all activated from procaspase 3 and procaspase 9 to their active form. Based on molecular modeling and Western blot protein expression results, it can be confirmed that kadsuric acid interacts with the DNA of PANC-1 cancer cells through apoptosis targeting PARP1. This can guide future studies to search for active ingredients from Morus alba as well as to use kadsuric acid as a precursor for semi-synthetic production of active substances for cancer treatment. 5. Conclusion In this study, we demonstrated that exhibited potent cytotoxic activity against human PANC-1 cancer cells. This compound induced apoptosis in human pancreatic cancer cells through caspase signaling pathway. For underlying mechanism insights, kadsuric acid displayed a strong binding affinity to PARP1 at -9.3 kcal/mol. The molecular dynamics simulation also showed that the complex between kadsuric acid and PARP1 is stable over 200 ns. Western blot analysis also confirmed that kadsuric acid reduced PARP1 expression in PANC-1 cells. Thus, it can be determined that kadsuric acid can interact with the DNA of human pancreatic cancer cells through caspase and PARP1. These results can guide future research on kadsuric acid targeting PARP1 as a natural anti-cancer substance. Declarations Acknowledgments The authors are thankful to School of Medicine and Pharmacy and VN-UK Institute for Research and Executive Education, The University of Danang for providing the infrastructure and facilities to perform this study. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Ethics approval Not required. Authors’ contribution statements TKN, TTT, PCHT, and LHDP : methodology, resources, software, investigation, writing-original draft; MHT, PTVP : conceptualization, methodology, supervision, writing-original draft, writing-review, and editing. References Abraham, M. J., Murtola, T., Schulz, R., Páll, S., Smith, J. C., Hess, B., & Lindah, E. (2015). 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4328289","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":306021702,"identity":"c9b81e22-ac17-439e-9e7b-ab85482e981a","order_by":0,"name":"Tan Khanh Nguyen","email":"","orcid":"","institution":"Dong A University","correspondingAuthor":false,"prefix":"","firstName":"Tan","middleName":"Khanh","lastName":"Nguyen","suffix":""},{"id":306021703,"identity":"0c63eb11-dc43-49d8-9f0f-e0daa2db5ebb","order_by":1,"name":"Manh Hung Tran","email":"","orcid":"","institution":"The University of Danang","correspondingAuthor":false,"prefix":"","firstName":"Manh","middleName":"Hung","lastName":"Tran","suffix":""},{"id":306021704,"identity":"ce4d9d55-5238-4d04-9fb8-e8e918fbb826","order_by":2,"name":"Tan Trung Truong","email":"","orcid":"","institution":"Dong Nai University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Tan","middleName":"Trung","lastName":"Truong","suffix":""},{"id":306021705,"identity":"0a482cde-4795-4a53-bdd7-3d38fc3cacaf","order_by":3,"name":"Long-Hung Dinh Pham","email":"","orcid":"","institution":"University of Medicine and Pharmacy at Ho Chi Minh City","correspondingAuthor":false,"prefix":"","firstName":"Long-Hung","middleName":"Dinh","lastName":"Pham","suffix":""},{"id":306021706,"identity":"a9d5246c-0ea0-4d6e-8e17-a8fc18bdc2ef","order_by":4,"name":"Phu Chi Hieu Truong","email":"","orcid":"","institution":"The University of Danang","correspondingAuthor":false,"prefix":"","firstName":"Phu","middleName":"Chi Hieu","lastName":"Truong","suffix":""},{"id":306021707,"identity":"402b4890-208d-4f6a-81e0-e08538fc2b68","order_by":5,"name":"Phu Tran Vinh Pham","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0002-4359-4924","institution":"VN UK Institute for Research and Executive Education","correspondingAuthor":true,"prefix":"","firstName":"Phu","middleName":"Tran Vinh","lastName":"Pham","suffix":""}],"badges":[],"createdAt":"2024-04-26 08:30:32","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4328289/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4328289/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s43450-024-00588-7","type":"published","date":"2024-09-11T15:58:24+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":57779799,"identity":"c7ea18da-e7b8-4d28-b16c-28653153b719","added_by":"auto","created_at":"2024-06-05 14:39:37","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":234157,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of kadsuric acid on PANC-1 cells. (\u003cstrong\u003ea\u003c/strong\u003e) Chemical structure of kadsuric acid, (\u003cstrong\u003eb\u003c/strong\u003e) Activation of kadsuric acid on caspase-3, (\u003cstrong\u003ec\u003c/strong\u003e) PARP degradation protein expression, and (\u003cstrong\u003ed\u003c/strong\u003e) caspase-3 and caspase-9 by kadsuric acid in PANC-1.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4328289/v1/d58a1bf5e6bba12d72b6cb67.png"},{"id":57780325,"identity":"91646426-5cb3-4024-8f8e-341c2f004608","added_by":"auto","created_at":"2024-06-05 14:47:37","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":340406,"visible":true,"origin":"","legend":"\u003cp\u003eBinding diagram of kadsuric acid with PARP1. (\u003cstrong\u003ea\u003c/strong\u003e) Docking pose (yellow) and crystal pose (green) of reference compound at the crystal structures of PARP1, and \u003cstrong\u003e(b) \u003c/strong\u003eInteraction between kadsuric acid and PARP1.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4328289/v1/cfeccce1fe2427bb86dc9464.png"},{"id":57779803,"identity":"227ffc1d-521f-442c-b0a4-6e59636dfa72","added_by":"auto","created_at":"2024-06-05 14:39:37","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":337562,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eHighest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) profiles, and (\u003cstrong\u003eb\u003c/strong\u003e) molecular electrostatic potential (MEP) surface of Kadsuric acid at the B3LYP/6-311G(d,p) level of theory.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4328289/v1/70fe5db9665c48a65551cc8f.png"},{"id":57780326,"identity":"e18f7964-0373-4ea5-b162-fa1ccb4b07d9","added_by":"auto","created_at":"2024-06-05 14:47:37","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":241546,"visible":true,"origin":"","legend":"\u003cp\u003eMolecular dynamic simulation of kadsuric acid and PARP1 complex during 200 ns. (\u003cstrong\u003ea\u003c/strong\u003e) RMSD, (\u003cstrong\u003eb\u003c/strong\u003e) RMSF, (\u003cstrong\u003ec\u003c/strong\u003e) radius of gyration, (\u003cstrong\u003ed\u003c/strong\u003e) number of hydrogen bonds (\u003cstrong\u003ee\u003c/strong\u003e) SASA (\u003cstrong\u003ef\u003c/strong\u003e) average center of mass distance\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4328289/v1/fc352660ce0804f585288f65.png"},{"id":57779800,"identity":"655f3ed7-58fc-4947-b18f-4374df40fc39","added_by":"auto","created_at":"2024-06-05 14:39:37","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":179900,"visible":true,"origin":"","legend":"\u003cp\u003ePrincipal component analysis of PARP1 proteintrajectory during 200 ns. (\u003cstrong\u003ea\u003c/strong\u003e) Trajectory projection on largest eigenvectors, (\u003cstrong\u003eb\u003c/strong\u003e) Gibbs free energy landscape\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4328289/v1/691038ae7163f957ed61cc1c.png"},{"id":64619369,"identity":"24fb3db4-f048-454f-9551-a7f18e2dc152","added_by":"auto","created_at":"2024-09-16 16:14:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2283016,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4328289/v1/b4553b6e-f941-4555-bcdd-96e30a450b80.pdf"},{"id":57779806,"identity":"88c8abaf-09db-4956-9a08-c9ce6822447e","added_by":"auto","created_at":"2024-06-05 14:39:37","extension":"jpg","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":333272,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicabstract.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4328289/v1/7eed787974bd66bee6fb4127.jpg"},{"id":57779804,"identity":"4c70ae2c-ba44-4da1-a450-c61b00b113fd","added_by":"auto","created_at":"2024-06-05 14:39:37","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":210014,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-4328289/v1/b6209e8d2d83ab85ce713bc9.docx"}],"financialInterests":"","formattedTitle":"Apoptosis induction of kadsuric acid from Vietnamese Kadsura coccinea (Lem.) A. C. Smith in human pancreatic cancer cells: in vitro and in silico approach","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe advancement of computational biology has provided new opportunities for research in the development of medicines for the health sciences. In particular, \u003cem\u003ein silico\u003c/em\u003e techniques have become increasingly important in exploring the biological effects of potential active ingredients on the human body(Roy et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). \u003cem\u003eIn silico\u003c/em\u003e methods can be used to identify drug targets, evaluate interactions between compounds and proteins, perform pharmacokinetic analysis (ADME), and improve the drug properties of molecules (Nguyen, Nguyen, et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Nguyen, Tran, et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). These techniques have already proven to be highly effective in research and are expected to pave the way for new drug development projects (Nguyen, Tran, et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePoly ADP-ribose Polymerase 1 (PARP1) is an enzyme found in the nucleus that is important for regulating various cellular processes, including DNA repair, transcription, and chromatin remodeling (Dadheech et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Tao \u0026amp; Wu, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). It belongs to a family of enzymes called ADP-ribosyltransferases, which modify proteins by adding ADP-ribose units. In cancer treatment, inhibiting PARP1 prevents cancer cells from repairing DNA damage, ultimately leading to cell death. However, normal cells with functional DNA repair mechanisms can tolerate PARP1 inhibition (Li et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Several PARP1 inhibitors, including olaparib, rucaparib, niraparib, and talazoparib, have been approved for the treatment of various types of cancer, particularly ovarian and breast cancer, and have shown promising results in clinical trials (Li et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Tao \u0026amp; Wu, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eKadsura coccinea\u003c/em\u003e (Schisandraceae) is a climbing vine with a woody, brown-black stem, and smooth branches. It is found in high mountain areas with tropical or subtropical climates in India, Laos, Vietnam, and southern China (Pham Hoang Ho, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Folk experience shows that \u003cem\u003eK. coccinea\u003c/em\u003e is used as a tonic, activating blood, alleviating pain, and stimulating digestion. The fruits are edible when ripe, the seeds are sometimes used as a replacement for the medicine \u003cem\u003eSchisandra chinensis\u003c/em\u003e (Y. Yang et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). According to previous studies, the main chemical components of \u003cem\u003eK. coccinea\u003c/em\u003e are lignan and triterpenoid compounds. Many lignans have been found in this plant with dibenzocyclooctadiene structures such as kadsutherin, isokadsuranin, kadsurins (Long et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Woo et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Y. Yang et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Q. J. Zhao et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; T. Zhao et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), among them, some substances could inhibit the formation of nitric oxide (NO) during the inflammatory process and anti-cancer (Daniyal et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Lu et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Y. Yang et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Q. J. Zhao et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; T. Zhao et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The triterpenoids were isolated from this plant (Hu et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Liang et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Xu et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Y. Yang et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), and were showed to inhibit the growth of human leukemia cancer cells (Daniyal et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Tram et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). In addition, extracts of \u003cem\u003eK. coccinea\u003c/em\u003e exhibited hepatoprotective effects in mice, anti-gastric cancer, and other several pharmaceutical effects (Daniyal et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Y. P. Yang et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2021\u003c/span\u003ea). However, in Vietnam, there are very few scientific studies on the chemical composition and biological effects of \u003cem\u003eK. coccinea\u003c/em\u003e (Ban et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOur previous research showed that \u003cem\u003eK. coccinea\u003c/em\u003e is a valuable plant source capable of inhibiting the growth of many carcinoma cell lines, including two pancreatic cancer cell lines PANC-1 and MIA PACA. In addition, we isolated and determined the chemical structure of several lanostane triterpenoids from the leaves of \u003cem\u003eK. coccinea\u003c/em\u003e including kadsuric acid as a main phytochemical component (Le et al., 2018). In this study, we reported the cytotoxic effects of this natural compound on PANC-1 cancer cells via the integration of \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein silico\u003c/em\u003e methods.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Kadsuric acid sample\u003c/h2\u003e \u003cp\u003eKadsuric acid: White amorphous powder; [α]\u003csup\u003e24\u003c/sup\u003e\u003csub\u003eD\u003c/sub\u003e + 68\u0026ordm; (c 0.25, MeOH); EI-MS m/z 470.3 [M]\u0026thinsp;+\u0026thinsp;C\u003csub\u003e30\u003c/sub\u003eH\u003csub\u003e46\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e. \u003csup\u003e1\u003c/sup\u003eH-NMR (CDCl\u003csub\u003e3\u003c/sub\u003e, 400 MHz) and \u003csup\u003e13\u003c/sup\u003eC-NMR (CDCl\u003csub\u003e3\u003c/sub\u003e, 100 MHz) were described in detail in our previous publication (Le et al., 2018). The chemical structure of kadsuric acid is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Equipment and instruments used in cell toxicity experiments\u003c/h2\u003e \u003cp\u003eEquipment and instruments used in cell toxicity experiments included 96-well plates (SPL Life Sciences, Korea), centrifuge tubes 1.5 mL, 2.0 mL, surface treated culture plate, multichannel pipette (Isolab, Germany), hemocytometer, cell counting chamber (improved Neubauer); Panasonic CO\u003csub\u003e2\u003c/sub\u003e incubator (MCO19AIC), ELISA reader machine (ELISA Bio-Rad machine, USA), autoclave sterilization, and drying cabinet. Chemicals used in evaluating the cytotoxic effects on cancer cells include Dulbecco's modified Minimum Essential Medium (DMEM), Roswell Park Memorial Institute (RPMI) 1640 Medium (Gibco, USA), phosphate buffer saline (PBS) (Gibco, USA), fetal bovine serum (FBS) (Gibco, USA); MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (DUCHEFA biochemie, Netherlands).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Cancer cells and cell cultures\u003c/h2\u003e \u003cp\u003eThe human pancreatic cancer PANC-1 cell lines were provided by Prof. Jeong-Hyung Lee, Kangwon National University, Korea. The cell lines were cultured at 37\u0026deg;C in DMEM supplemented with 10% Fetal Bovine Serum (FBS), 100 U/mL penicillin, and 100 mcg/mL streptomycin in a 5% CO\u003csub\u003e2\u003c/sub\u003e incubator for 48 h.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Evaluate caspase 3 activation\u003c/h2\u003e \u003cp\u003eCaspase-3 is an enzyme in the caspase family (cysteine-aspartic protease enzymes) that normally exists as an inactive precursor in cells. When this enzyme is activated, it initiates the process of cell death (apoptosis) by cleaving a protein (substrate) that specifically targets the amino chain DEVD (Asp-Glu-Val-Asp). Ac-DEVD-AFC is a substrate labeled with a fluorescent molecule, that is cleaved by caspase-3 at the bond between D and AFC (7-amino-4-trifluoromethylcoumarin) as entering a cell, thereby releasing the fluorescent AFC that can be quantified by fluorescence spectrophotometer (Tuan et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Vo et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Cancer cells were treated with reagents at various concentrations from 1 to 30 \u0026micro;M. After incubation for 12, 24, and 48 h, the cells were harvested, washed with cold PBS, and then lysed in a lysis buffer at room temperature for 10 min, then cooled and added to a test buffer in each well, incubated at 37\u0026deg;C for 1 h. The fluorescence intensity was measured by stimulation at 370 nm and emission at 505 nm using a Twinkle LB970 machine (Berthold Technologies, Germany). The sample containing only cancer cells without compound perturbation was used as the control group. The extent of caspase-3 enzyme induction was determined by the ratio of the fluorescence intensity of the test sample to that of the control sample. The results were processed using GraphPad Prism 3.03 software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Molecular docking\u003c/h2\u003e \u003cp\u003eThe crystal structures of PARP1 (PDB ID: 5WS1) were downloaded from Protein Data Bank (RCSB PDB). The preparation process was following our previous study (Tran et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The process of removing unrelated co-crystallized ligands and water molecules was done by using the Discovery Studio 2020 Client software. After that, the protein structure was minimized using SwissPDB. Then, molecular hydrogens and Kollman charges were added to the protein by the AutoDock Tool software version 1.5.6 (Morris et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). The active site of protein was also selected according to the interactive site of co-crystallized ligand and protein. The grid box site was set at 18.75 \u0026times; 18.75 \u0026times; 18.75 for x,y and dimensions. For ligand preparation, the 3D structures of the compound were downloaded from the academic structural library PubChem. Then, Open Babel software was used to convert to .pdbqt format for docking process. The application Autodock Vina version 1.2.3 was implemented to carry out the molecular docking of PARP1 (Eberhardt et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The docking score was presented in kcal/mol.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Density Functional Theory (DFT)\u003c/h2\u003e \u003cp\u003eThe DFT/B3LYP method (Becke, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1993\u003c/span\u003e) with a 6-311G(d,p) basis set in the gas phase was used in these calculations. The highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) energy have been computed. Furthermore, the energy gap (Eg) and several other important parameters, which as ionization potential (\u003cem\u003eIP\u003c/em\u003e), electron affinity (\u003cem\u003eEA\u003c/em\u003e), electronegativity (χ), chemical hardnesses (η), electronic chemical potential (\u0026micro;), chemical softness (S), and electrophilicity (ω) were calculated by using Eqs.\u0026nbsp;(1)-(8) (Medoro et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEg\u0026thinsp;=\u0026thinsp;E\u003csub\u003eLUMO\u003c/sub\u003e - E\u003csub\u003eHOMO\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(1)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eIP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;-\u0026thinsp;E\u003csub\u003eHOMO\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(2)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eEA\u003c/em\u003e\u0026thinsp;=\u0026thinsp;-\u0026thinsp;E\u003csub\u003eLUMO\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(3)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eχ = -(E\u003csub\u003eLUMO\u003c/sub\u003e + E\u003csub\u003eHOMO\u003c/sub\u003e)/2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(4)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eη = (E\u003csub\u003eLUMO\u003c/sub\u003e - E\u003csub\u003eHOMO\u003c/sub\u003e)/2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(5)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026micro; = -χ = (E\u003csub\u003eLUMO\u003c/sub\u003e + E\u003csub\u003eHOMO\u003c/sub\u003e)/2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(6)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eS\u0026thinsp;=\u0026thinsp;1/2η\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(7)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eω\u0026thinsp;=\u0026thinsp;\u0026micro;\u003csup\u003e2\u003c/sup\u003e/2η\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(8)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe molecular\u0026rsquo;s electrophilic/nucleophilic reactive location was determined using molecular electrostatic potential (MEP). All calculation was carried out with the Gaussian 16 program (Frisch, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) and data from Gaussian output files were visualized with the help of the program GaussView 6.0 (Dennington, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Molecular dynamic simulation\u003c/h2\u003e \u003cp\u003eThe protocol of simulation was performed following our previous study (Bich et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Molecular dynamics simulations were performed using GROMACS 2020.4 in order to further investigate the stability of the ligand in complex with PARP1 (Abraham et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Pronk et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The complex\u0026rsquo;s topology was prepared under the CHARMM36 forcefield and solvated in a truncated octahedral box containing TIP3P water molecules using the CHARMM-GUI server (Jo et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). To ensure a fully stable system, the objective values of 300 K temperature and 1 bar pressure were reached. Finally, the molecular dynamics simulation was run for 200 ns for the complex.\u003c/p\u003e \u003cp\u003ePost-simulation principal component analysis (PCA) was implemented using in-built GROMACS modules \u003cem\u003egmx covar\u003c/em\u003e, \u003cem\u003egmx anaeig\u003c/em\u003e, and \u003cem\u003egmx sham\u003c/em\u003e. First, the covariance matrix of protein atoms' trajectories was calculated and diagonalized using \u003cem\u003egmx covar\u003c/em\u003e. Trajectories were then projected on the two largest eigenvectors (named PC1 and PC2) for visualization and further predicted of Gibbs free energy landscape using \u003cem\u003egmx sham\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Calculate Binding Free Energy (MM/PBSA Calculations)\u003c/h2\u003e \u003cp\u003eMolecular mechanics Poisson\u0026ndash;Boltzmann surface area (MM/PBSA) has been regarded as a competitive method to estimate the relative binding free energies for the compound and to evaluate the relative stability of the apoprotein. In this investigation, the MOLAICAL script was used to implement the MM/PBSA model to measure the relative binding free energy (Bai et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Western Blot assay\u003c/h2\u003e \u003cp\u003eProtein-to-protein hybridization (antigen to antibody), antigenic protein is detected by coloration or fluorescence reaction. In Western Blot, a protein mixture was separated by SDS-PAGE electrophoresis. Protein lines were transferred onto the hybrid membrane and individual protein bands were detected by immersing the membrane with primary and secondary antibodies labeled with enzymes or radiolabeled specific for the protein of interest. If the protein of interest is bound by a radiolabeled antibody, its position on the spots can be detected by placing the film on an X-ray film. In our experiment, the cells were incubated with the inhibitor for 24 h at 37\u0026deg;C. Cell fragments were immersed in a lysis buffer containing a mixture of protease inhibitors, and centrifuged to remove the precipitate. Measure the concentration of the supernatant. This protein supernatant was separated by polyacrylamide electrophoresis with SDS (SDS-PAGE) and then transferred to a PVDF membrane filter (Bio-Rad, USA). Place the membrane in 5% nonfat milk powder in Tris buffer containing 0.1% Tween-20 (TBS-T) at 4\u0026deg;C overnight and incubate with the primary antibody at room temperature. Membranes were washed three times with TBS-T and permeabilized with HRP-labeled secondary antibodies (Horse-radish peroxidase), incubated at room temperature for 1.5 h, and then washed three times with TBS-T. Detection of immunological proteins and X-rays. Protein was normalized by repulsing the same membrane with the detection of anti-β-actin antibodies, the membranes were immersed in a rejection buffer at room temperature for 20 min (Vo et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Activation of enzyme caspase 3 and western blotting analysis\u003c/h2\u003e \u003cp\u003eThe chemical structure of Kadsuric acid is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea. This compound is a 3,4 seco-lanostane triterpene and is present in large amounts from \u003cem\u003eKadsura coccinea\u003c/em\u003e species (Le et al., 2018; Liang et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Y. Yang et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Y. P. Yang et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2021\u003c/span\u003eb). Kadsuric acid exhibited the cytotoxic activity against PANC-1 with an IC\u003csub\u003e50\u003c/sub\u003e value of 14.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8 \u0026micro;M. To investigate the cytotoxicity mechanism, this substance was consequently selected for testing on caspase-3 activation and Western Blot analysis on PANC-1 cells. PANC-1 cells were incubated with kadsuric acid (1\u0026ndash;30 \u0026micro;M) at 37\u0026deg;C with caspase-3 substrate (Ac-DEVD-AFC) for 1h. The fluorescence intensity of the cell lysates was measured to determine caspase-3 activity. The negative control sample contained DMSO 0.1%. Data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD of the results from three independent experiments (*p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; **p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Then, cells were treated with kadsuric acid (1\u0026ndash;30 \u0026micro;M) for 48 h. Protein 50 \u0026micro;g/lane from cell lysates were electrophoresed on SDS-PAGE gels and then transferred to total blot PVDF membranes. β-actin was used as an internal control, (\u0026ndash;), 0.1% DMSO-treated cells.\u003c/p\u003e \u003cp\u003eActivation of caspase-3 leads to the activation of other caspases, such as caspase-6 and caspase-7, which cleave multiple protein structures and regulate protein conformational transformation, which is crucial for cell survival and maintenance. Among caspase family enzymes, caspase-3 was identified as the most important enzyme (executioner enzyme) and is activated by both endogenous and exogenous pathways in the cell (Brentnall et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Therefore, activating the caspase-3 enzyme will lead to cell death, which is the main mechanism of current cancer treatments. The results showed that kadsuric acid activated caspase-3, increasing the level of enzyme cleavage by 1\u0026ndash;2 times after 12 and 24 h, and increasing the cleavage, the activation of caspase-3 by more than 3\u0026ndash;4 times compared to the negative control PANC-1 cells that were not cleaved (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). These results suggest that kadsuric acid can increase the activation of caspase-3 in the PANC-1 cell line, depending on the concentration and incubation time in the cell culture medium.\u003c/p\u003e \u003cp\u003eIn next process, kadsuric acid was validated for the direct interaction with PAPR1, which is the target protein in caspase signaling. We also used cleaved poly (ADP-ribose) polymerase (cleaved poly (ADP-ribose) polymerase, PARP) treated with kadsuric acid (1\u0026ndash;30 \u0026micro;M) and the protein expression was expressed by western blot analysis. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec, kadsuric acid induced transformation of PARP inducing the cleaved form of PARP (cleaved PARP) on PANC-1 cells after 48 h. PARP is a protein that plays an important role in helping damaged cells repair themselves. For normal cells, PARP has a role in self-repairing and repairing damage when DNA is broken during the repair process. However, for cancer cells, PARP has a role in the self-repairing of damaged DNA in cancer cells when exposed to foreign agents, which allows cancer cells to be resistant to agents such as drugs or chemotherapy and continue to divide (Alemasova \u0026amp; Lavrik, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Therefore, for cancer cell lines, drugs that inhibit the activity of PARP protein can slow or stop tumor growth. In this experiment, kadsuric acid played an inhibitory role and changed the structure of PARP into a cleaved protein, whereby PARP would not be able to perform its repair function to restore the damaged DNA of cancer cells. This is the main mechanism of PARP pathway anticancer drugs. Analysis of the cleaved PARP protein content showed that kadsuric acid (1\u0026ndash;30 \u0026micro;M) modifies the PARP protein composition and interferes with PARP's ability to repair itself, inducing PANC-1 cell apoptosis in a dependent manner of concentration.\u003c/p\u003e \u003cp\u003eTo confirm the downstream activity of PAPR1 inhibitor, the effect of kadsuric acid (1\u0026ndash;30 \u0026micro;M) on caspase-3 and caspase-9 activation was further examined in the PANC-1 cancer cell line (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). Typically, caspase-3 exist as inactive precursors of 32 kDa (procaspase-3). When activated, caspase-3 converts to an activated form (activated caspase-3) and causes the cleavage of many DNA fragments in cancer cells, which can induce cell death by initiating the apoptosis pathway through the cleavage of DNA and proteins into distinct apoptotic fragments. While caspase-3 is considered the executioner of cell death, caspase-9 also participates in the apoptosis process of cells, this enzyme is encoded by the CASP9 gene, which is the initiator of cancer cell death. Our result showed that kadsuric acid (1\u0026ndash;30 \u0026micro;M) activated procaspase-3 and procaspase-9 to their active forms. Accordingly, when increasing the concentration of these fractions, the expression of caspase-3 and caspase-9 enzyme proteins also increased. This could lead to the conclusion that both segments induce apoptosis of PANC-1 cells. From these findings, we demonstrated that kadsuric acid induced apotosis through activating capase-3 pathway by interacting directly PARP1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Molecular docking analysis\u003c/h2\u003e \u003cp\u003eTo further investigate the interactions between kadsuric acid and PARP1, molecular docking was applied. PARP-1 enzyme binds to DNA via transfer chains of ADP-ribosyl moieties (PARs) and two zinc finger motifs from nicotinamide-adenine-dinucleotide (NAD+) to chromatin-associated acceptor proteins. This enzyme plays an important role in promoting DNA repair in cancer cells (Houtgraaf et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Therefore, the search for compounds with inhibitory activity of PARP1 is considered an effective approach for the development of cancer drugs. In this study, kadsuric acid was docked at the catalytic site of PARP1 to evaluate the binding ability and predict the anticancer mechanism of this compound. The 3D structure of domain of PARP1 (PDB ID: 5WS1) was selected for screening with 352 amino acids from His660 to Thr101. The grid box was set to cover the active site of PARP1 following the interactive site of co-crystalized ligand. To confirm the reliability of the chosen protein structure and docking procedure, the 2-[(3R)-3-azanylpyrrolidin-1-yl] carbonyl-1H-benzimidazole-4-carboxamide co-crystal ligand was subjected to re-docking. This involved analyzing the root mean square deviation (RMSD) between the docking pose and the crystal pose. The findings showed that the docking position had comparable conformation and orientation to the crystal pose, with an RMSD value of 0.525 \u0026Aring; (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). The results revealed kadsuric acid had docking score of \u0026ndash; 9.3 kcal/mol, which was better than the docking score of the reference compound, \u0026minus;\u0026thinsp;8.6 kcal/mol. The results are presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The crystal ligand showed interaction with PARP1 with two hydrogen bonds at His862, Ser904 residues and three hydrophobic interactions at Tyr907, Ala898, and Gly863 residues. Meanwhile, kadsuric acid was found to interact with enzyme with three hydrogen bonds at Ser864, Asp770, and Glu763 residues and six hydrophobic interactions at His862, Leu877, Ile872, Tyr896, Tyr907, and Met890 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). In particular, acid residues of the triad, His862, and Tyr896, are required for binding of NAD+, while Glu988, besides substrate positioning, is also critical for catalysis. Notably, similar to the reference compound, kadsuric acid showed interactions with key amino acid residues of the donor site His862, and Tyr896 that play an important role in PARP1 activity (Alemasova \u0026amp; Lavrik, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDocking results of candidates towards PARP1 protein\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCompound\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDocking score (kcal/mol)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHydrogen bond\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHydrophobic interaction\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKadsuric acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u0026minus;\u0026thinsp;9.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSer864, Asp770, Glu763\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHis862, Leu877, Ile872, Tyr896, Tyr907, Met890\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2-[(3R)-3-azanylpyrrolidin-1-yl] carbonyl-1H-benzimidazole-4-carboxamide\u003csup\u003ea)\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u0026minus;\u0026thinsp;8.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHis862, Ser904\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTyr907, Ala898, Gly863\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003e\u003csup\u003ea)\u003c/sup\u003eReference compound.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Density Functional Theory (DFT)\u003c/h2\u003e \u003cp\u003eThe frontier molecular orbitals (HOMO and LUMO) of the optimized structure (Kadsuric acid) are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea. Calculated geometric parameters (\u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e) of Kadsuric acid compound are given in \u003cb\u003eSupplementary\u003c/b\u003e. Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows detailed quantum chemical parameters of Kadsuric acid compound by DFT/B3LYP/6-311G(d,p) method. The computed energy value of HOMO (E\u003csub\u003eHOMO\u003c/sub\u003e) is -6.370 eV and the energy of LUMO (E\u003csub\u003eLUMO\u003c/sub\u003e) is -0.128 eV. The energy gap (Eg) between HOMO and LUMO of the Kadsuric acid molecule is 6.242 eV, lying on the transition of an insulator (\u0026gt;\u0026thinsp;9 eV) and a semiconductor (\u0026lt;\u0026thinsp;3.2 eV). This energy level can be interpreted as favorable to intermolecular binding to protein structures (Van Chen et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Additionally, the reactivity descriptors properties such as electronegativity (χ), chemical hardnesses (η), electronic chemical potential (\u0026micro;), chemical softness (S), and electrophilicity (ω) were also calculated. Furthermore, molecular electrostatic potential (MEP) was also calculated at B3LYP/6-311G(d,p) level of theory to forecast the electrophilic/nucleophilic reactive sites of Kadsuric acid. In this, the region having the negative potentiality (deep red) over oxygen atoms (O2 and O4) and positive potentiality (deep blue) over hydrogen atoms (H76 and H80) which suggests for the electrophilic and nucleophile attack the respected region, respectively, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb. Altogether, the findings from quantum chemistry investigations suppose Kadsuric acid has good interactions with protein.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe calculated quantum chemical parameters of the investigated compound by using DFT\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eParameters\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKadsuric acid\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eB3LYP/6-311G(d,p) (eV)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEnergy of HOMO (E\u003csub\u003eHOMO\u003c/sub\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-6.370\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEnergy of LUMO (E\u003csub\u003eLUMO\u003c/sub\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-0.128\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEnergy gap (E\u003csub\u003eg\u003c/sub\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6.242\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eReactivity descriptors\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIonization potential \u003cem\u003e(IP)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6.370\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eElectron affinity \u003cem\u003e(EA)\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.128\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eElectronegativity (χ)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3.249\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChemical hardnesses (η)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3.121\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eElectronic chemical potential (\u0026micro;)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e-3.249\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChemical softness (S)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.160\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eElectrophilicity (ω)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1.692\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Molecular dynamics simulation\u003c/h2\u003e \u003cp\u003eSmall molecules interacting with protein surfaces can induce large tertiary structure changes, which can be exploited in drug design improvements. The main advantage of molecular dynamics simulation is its ability to identify the stability and flexibility of the protein-ligand complex. This method was used to precisely evaluate the thermodynamics and kinetics of drug-enzyme binding. The docking pose with the highest docking score was selected for the simulation process. The 200 ns trajectories of the complex between kadsuric acid and PARP1 were analyzed to evaluate the stability of the ligands and protein.\u003c/p\u003e \u003cp\u003eThe findings demonstrated that the structure maintained its stability over the course of the simulation with fluctuation within the range of 1 \u0026Aring; (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). The RMSF results also showed that the ligand stabilized a number of interaction-related residues at the active site (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). The distribution of a protein\u0026rsquo;s atoms along its axis is referred to as the radius of gyration (Rg) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). The solvent-accessible surface area (SASA) facilitates the visualization of the protein\u0026rsquo;s conformational change over ligand binding. Both values fluctuate lightly with values of 0.8 \u0026Aring; (for Rg) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec) and 200 \u0026Aring;\u003csup\u003e2\u003c/sup\u003e (for SASA) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee), thus confirming the stability of the protein and the complex. It is highlighted that the total number of hydrogen bonds formed between ligand and protein during 200 ns was also calculated. During the simulation process, the ligand almost always formed 2 to 3 hydrogen bonds with the protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). The average center-of-mass distance between the ligand and the protein during simulation time is shown in (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef). The result indicated the ligands showed a stable distance with less than 1.5 \u0026Aring; of fluctuation.\u003c/p\u003e \u003cp\u003eTrajectory PCA analysis revealed a relatively smooth and well-clustered transition of states during 200 ns (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The Gibbs free energy landscape also ranged moderately from 0\u0026ndash;9.43 kJ/mol, with a majority in the low-energy zone (colored green), thus once again suggesting favourable conditions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Binding free energy\u003c/h2\u003e \u003cp\u003eAt the final stage of simulation, the binding free energy of kadsuric acid further explained the potency of kadsuric acid. The different energy components contributing to binding energy are shown in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. These results confirmed the molecular docking results and demonstrated that kadsuric acid can maintain strong binding with PARP1.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComponents of binding free energy calculation\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCompound\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVan der Waal energy (kJ/mol)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eElectrostatic energy (kJ/mol)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePolar solvation energy (kJ/mol)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSASA energy (kJ/mol)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eBinding energy (kJ/mol)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKadsuric acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-166.690 \u0026plusmn; 16.894\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-161.417 \u0026plusmn; 32.083\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e218.225 \u0026plusmn; 22.584\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-22.607 \u0026plusmn; 1.502\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-132.489 \u0026plusmn; 28.539\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eApoptosis is a form of cell death (programmed death) that occurs under pathological conditions in organisms and contributes to cell replacement, tissue regeneration, and elimination of damaged cells under adverse normal conditions (Brentnall et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Chaudhry et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Most cancers are caused by abnormal changes in the genetic material of cells during development. Abnormalities may be caused by the effects of carcinogenic chemicals such as cigarette smoke, radiation, chemicals, or infectious agents. Genetically, cancer is a random error in DNA replication or is inherited, so it can be present in all cells from birth (Houtgraaf et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Yam et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In fact, a complex interaction between carcinogens and the host genome may account for cancer development immediately after cell exposure to a stimulus. There are many types of activation to initiate apoptosis, including activation of the caspase enzyme system (Brentnall et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Yosefzon et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Scientists have found that one of the key mechanisms is that caspase enzymes are activated prior to mitochondrial changes (Brentnall et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Studies have shown that activation of the enzyme caspase-9 followed by activation of caspase-3 initiates apoptosis. Caspase enzymes are also able to cleave many other structural forms, including cytoskeletal, nuclear, and proteomic factors, lamin, actin, and conjugated ADP-ribose (PARP) (Brentnall et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The cleavage of these proteins is associated with cellular morphological alterations that have been demonstrated extensively during apoptosis (Chaudhry et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThere were many studies on the application of natural medicinal herbs and active ingredients from plants to destroy cancer cells and support the treatment of cancers (Cragg et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2005\u003c/span\u003e)(Kinghorn et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Some of the commonly used cancer drugs in the podophyllins group are etoposide and teniposide. The two main components of \u003cem\u003eCatharanthus roseus\u003c/em\u003e, vinblastine, and vincristine, have been shown to have the ability to kill cancer cells. Another important compound is paclitaxel (Taxol) (Cragg et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Gordaliza, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Taxol found in the bark of the \u003cem\u003eTaxus brevifolia\u003c/em\u003e was used in the treatment of ovarian, breast, lung, pancreatic cancers (Cragg et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Kinghorn et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn our experiment, the effect of kadsuric acid on the activation of caspase 3 showed that PANC-1 cancer cells may have been killed within 48 h when treated with kadsuric acid. Normally, caspase 3 exists as an inactive precursor with a size of 32 kDa (procaspase 3) (Brentnall et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Caspase 3 was converted to an activated form and caused the cleavage of multiple segments of cancer cell DNA, which can induce cell death by initiating apoptosis through the cleavage of DNA proteins into heterozygous compounds. While caspase 3 is considered a cell killer, caspase 9, another cysteine-aspartic acid protease, is also involved in cell apoptosis by encoding the CASP9 gene, which is the initiator of the death cells (Brentnall et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Yosefzon et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In this study, we discovered that kadsuric acid from \u003cem\u003eK. coccinea\u003c/em\u003e also inhibited the growth and development of PANC-1 pancreatic cancer cells. Kadsuric acid is a substance with the structure 3,4 seco-lanostane triterpene, this secondary metabolite showed to interact with PARP1, PARP1 was then cleaved by caspase 3 into an N-terminal 25 kDa and a C-terminal 85 kDa. The PARPs are a family involved in catalyzing the transport of ADP-ribose to target proteins. PARPs play important roles in various cellular processes, including modulation of chromatin structure, transcription, replication, recombination, and DNA repair. The role of PARP proteins in DNA repair is of particular interest, with the task of detecting errors in the recombination mechanism, PARP repairs cellular DNA to maintain the ability to recombine and divide. Most of the PARP inhibitors used in cancer therapy follow this mechanism.\u003c/p\u003e \u003cp\u003eOur study showed that kadsuric acid showed the ability to activate the caspase enzyme system consisting of 2 types of cysteine-aspartic acid proteases, caspase 3 and caspase 9. Through western blot protein expression, these enzymes were all activated from procaspase 3 and procaspase 9 to their active form. Based on molecular modeling and Western blot protein expression results, it can be confirmed that kadsuric acid interacts with the DNA of PANC-1 cancer cells through apoptosis targeting PARP1. This can guide future studies to search for active ingredients from \u003cem\u003eMorus alba\u003c/em\u003e as well as to use kadsuric acid as a precursor for semi-synthetic production of active substances for cancer treatment.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn this study, we demonstrated that exhibited potent cytotoxic activity against human PANC-1 cancer cells. This compound induced apoptosis in human pancreatic cancer cells through caspase signaling pathway. For underlying mechanism insights, kadsuric acid displayed a strong binding affinity to PARP1 at -9.3 kcal/mol. The molecular dynamics simulation also showed that the complex between kadsuric acid and PARP1 is stable over 200 ns. Western blot analysis also confirmed that kadsuric acid reduced PARP1 expression in PANC-1 cells. Thus, it can be determined that kadsuric acid can interact with the DNA of human pancreatic cancer cells through caspase and PARP1. These results can guide future research on kadsuric acid targeting PARP1 as a natural anti-cancer substance.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors are thankful to School of Medicine and Pharmacy and VN-UK Institute for Research and Executive Education, The University of Danang for providing the infrastructure and facilities to perform this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot required.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contribution statements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTKN, TTT, PCHT, and LHDP\u003c/strong\u003e: methodology, resources, software, investigation, writing-original draft; \u003cstrong\u003eMHT, PTVP\u003c/strong\u003e: conceptualization, methodology, supervision, writing-original draft, writing-review, and editing.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAbraham, M. J., Murtola, T., Schulz, R., P\u0026aacute;ll, S., Smith, J. C., Hess, B., \u0026amp; Lindah, E. (2015). GROMACS: High performance molecular simulations through multi-level parallelism from laptops to supercomputers. \u003cem\u003eSoftwareX\u003c/em\u003e, \u003cem\u003e1\u0026ndash;2\u003c/em\u003e, 19\u0026ndash;25. https://doi.org/10.1016/J.SOFTX.2015.06.001\u003c/li\u003e\n \u003cli\u003eAlemasova, E. E., \u0026amp; Lavrik, O. I. (2019). Poly(ADP-ribosyl)ation by PARP1: reaction mechanism and regulatory proteins. \u003cem\u003eNucleic Acids Research\u003c/em\u003e, \u003cem\u003e47\u003c/em\u003e(8), 3811. https://doi.org/10.1093/NAR/GKZ120\u003c/li\u003e\n \u003cli\u003eBai, Q., Tan, S., Xu, T., Liu, H., Huang, J., \u0026amp; Yao, X. (2021). 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Cytotoxic dibenzocyclooctadiene lignans from Kadsura coccinea. \u003cem\u003eArchives of Pharmacal Research\u003c/em\u003e, \u003cem\u003e37\u003c/em\u003e(11), 1375\u0026ndash;1379. https://doi.org/10.1007/S12272-013-0186-3\u003c/li\u003e\n \u003cli\u003eZhao, T., Ma, C., \u0026amp; Zhu, G. (2021). Chemical composition and biological activities of essential oils from the leaves, stems, and roots of kadsura coccinea. \u003cem\u003eMolecules\u003c/em\u003e, \u003cem\u003e26\u003c/em\u003e(20). https://doi.org/10.3390/molecules26206259\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"revista-brasileira-de-farmacognosia","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"rbfa","sideBox":"Learn more about [Revista Brasileira de Farmacognosia](https://www.springer.com/journal/43450)","snPcode":"43450","submissionUrl":"https://www.editorialmanager.com/rbfa/default2.aspx","title":"Revista Brasileira de Farmacognosia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Kadsura coccinea, kadsuric acid, caspases, PARP1, pancreatic cancer, molecular modeling","lastPublishedDoi":"10.21203/rs.3.rs-4328289/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4328289/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eKadsuric acid, a major triterpenoid isolated from the leaves of Vietnamese \u003cem\u003eKadsura coccinea\u003c/em\u003e, exhibited potent cytotoxic effects in some human cancer cells. In this study, the effects of kadsuric acid on pancreatic cancer cells PANC-1 were investigated. The results showed that kadsuric acid exhibited dose-dependent cytotoxicity against PANC-1 with an IC50 value of 14.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8 \u0026micro;M. Kadsuric acid effectively activated caspase-3 by increasing the level of enzyme cleavage by 1\u0026ndash;2 times after 12 and 24 h, and by more than 3\u0026ndash;4 times compared to the negative control. In addition, this compound enhanced both two types of cysteine-aspartic acid proteases, including caspase-3 and caspase-9 through protein expressions. Western blot analysis also indicated that kadsuric acid reduced Poly [ADP-ribose] polymerase 1 (PARP1) expression in PANC-1 cells. For underlying mechanism insights, molecular modeling methods were applied to investigate the binding interaction between kadsuric acid and PARP1. Compared to the co-crystallized ligand, kadsuric acid displayed a stronger binding affinity (-9.3 kcal/mol). A molecular dynamics simulation showed that the complex is stable over 200 ns. Taken together, it can be determined that kadsuric acid can interact with the DNA of human pancreatic cancer cells through the intrinsic caspase/PARP-1 pathway. This study can guide future research on kadsuric acid as PARP1 inhibitor for cancer treatment.\u003c/p\u003e","manuscriptTitle":"Apoptosis induction of kadsuric acid from Vietnamese Kadsura coccinea (Lem.) A. C. Smith in human pancreatic cancer cells: in vitro and in silico approach","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-05 14:39:32","doi":"10.21203/rs.3.rs-4328289/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2024-05-28T05:59:29+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-05-23T15:31:25+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Revista Brasileira de Farmacognosia","date":"2024-05-20T05:14:10+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-05-02T08:27:27+00:00","index":"","fulltext":""},{"type":"submitted","content":"Revista Brasileira de Farmacognosia","date":"2024-04-29T23:14:55+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"revista-brasileira-de-farmacognosia","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"rbfa","sideBox":"Learn more about [Revista Brasileira de Farmacognosia](https://www.springer.com/journal/43450)","snPcode":"43450","submissionUrl":"https://www.editorialmanager.com/rbfa/default2.aspx","title":"Revista Brasileira de Farmacognosia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"ec6e9967-ce68-412e-b67d-de334b8b6f7b","owner":[],"postedDate":"June 5th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-09-16T16:06:22+00:00","versionOfRecord":{"articleIdentity":"rs-4328289","link":"https://doi.org/10.1007/s43450-024-00588-7","journal":{"identity":"revista-brasileira-de-farmacognosia","isVorOnly":false,"title":"Revista Brasileira de Farmacognosia"},"publishedOn":"2024-09-11 15:58:24","publishedOnDateReadable":"September 11th, 2024"},"versionCreatedAt":"2024-06-05 14:39:32","video":"","vorDoi":"10.1007/s43450-024-00588-7","vorDoiUrl":"https://doi.org/10.1007/s43450-024-00588-7","workflowStages":[]},"version":"v1","identity":"rs-4328289","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4328289","identity":"rs-4328289","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}