Study on Wangzaozin-A-Inducing Cancer Apoptosis and Its Theoretical Protein Targets

Abstract

Wangzaozin A is the most representative cytotoxic C-20-nonoxide compound of Isodon plants of Labiatae. The protein targets of the 2 isomers (X and X′) of Wangzaozin A are, respectively, extracted from target fishing dock Web site. Each isomer has 23 targets with good energy scores. The binding modes of each isomer and its targets are, respectively, analyzed by Dock. The energy score of the binding mode between the isomer X and the inositol-1(or 4)-monophosphatase is the smallest. The apoptosis, antiproliferative, and lethal effects of human gastric cancer cells induced by Wangzaozin A are assessed by trypan blue exclusion, hoechst33258 stain in SGC-7901, and flow cytometric measurement. The mechanism of Wangzaozin-A-inducing cancer apoptosis is analyzed. Wangzaozin A inhibits the growth of human gastric cancer SGC-7901 cell lines at the lower concentration (<4.0 µmol/L) and results in the lethal effect at the relative higher concentration (>8.0 µmol/L).

Introduction

Wangzaozin A ^1,2 is the most representative cytotoxic Isodon plant. There are 150 Isodon species in Labiatae all over the world. The 90 species that grow in China mainly contain ent-Kauranoid-diterpenoid, ursane-triterpenoid, and flavonoid compounds. Over 400 ent-Kauranoid-diterpenoid compounds have been separated from Isodon plants, ³ many of which have favorable physiological activity and serve as folk medicines. From 4 Isodon plants growing in Gansu, China, we have separated 19 ent-Kauranoid-diterpenoid compounds, which mainly contain C-20-nonoxide, C-20-oxide, and enmein compounds. Wangzaozin A is the most cytotoxic one in the active C-20-nonoxide compounds. ^1,2,4 The cytotoxicity results from the special structure of Wangzaozin A. It is significant to study the functional change in the proteins in cancer cells after treating the cells using the Wangzaozin A solution.

Drug target validation and identification are very significant for drug discovery and development. ⁵ Numerous technologies for addressing the targets have been developed. ⁵ Methods in genomics and proteomics, including bioinformatics analysis, are major tools for drug target identification. Chemical biology, which generally uses small molecules as probes to map genomic functions, is an emerging tool for drug target identification. ⁶ A computational method named target fishing dock (TarFisDock) ⁷ has been developed by using a reverse docking method for drug target identification, that is, discovering the potential binding protein candidates of active compounds (natural products or existing drugs). For example, the drug targets of natural product N-trans-caffeoyltyramine have been searched from drug target database by TarFisDock, and the computational clues have been verified by enzymatic assay as well as X-ray crystallography determination. ⁸

Apoptosis or programmed cell death is a physiological process that plays a critical role in the normal development and maintenance of tissue homeostasis by eliminating the infected, mutated, or damaged cells in multicellular organisms. The morphological characterizations of apoptotic cells are shrinkage, plasma membrane bubbling, chromatin condensation, and nuclear membrane breakdown. The formations of small vesicles from cell surface are also known as apoptotic bodies. ⁹ The apoptotic machinery in humans consists of a molecular network of a large number of proteins ^10,11 that regulate a cascade of events in the signaling, commitment, and execution of the apoptosis through multiple parallel pathways. For cancer cure, cancer chemotherapy is very efficient. Therefore, it is significant to study drug-induced apoptosis.

In this article, the protein targets of the 2 isomers (X and X′) of Wangzaozin A are, respectively, extracted from TarFisDock Web site. Each isomer has 23 targets with good energy scores. The binding modes of each isomer and its targets are, respectively, analyzed by Dock. Furthermore, the cancer cells are induced into apoptosis after treating by the Wangzaozin A solution. The apoptosis, antiproliferative, and lethal effects of human gastric cancer cells induced by Wangzaozin A are assessed by trypan blue exclusion, hoechst33258 stain in SGC-7901, and flow cytometric measurement.

Materials and Methods

The Crystal Structure of Wangzaozin A

Wangzaozin A (C₂₀H₃₀O₄) is separated from the Isodon excisoides and the Isodon racemosa (Hemsl) Hara growing in Gansu, China. The crystal structure of Wangzaozin A has been determined by single-crystal X-ray diffraction analysis. ² The 2 isomers (Figure 1A, X and X′) of Wangzaozin A have small differences in bond lengths and angles. Three 6-membered rings of each isomer are in a chair-like conformation, and one 5-membered ring is in a twist envelope-like conformation. The crystal is in an orthorhombic crystal system. The accurate lattice constants of the space group P2₁2₁2₁ are as follows: a = 0.66485(8) nm, b = 2.5796(4) nm, c = 1.0473(2) nm, and Z = 4. The molecules form extensive networks through the intramolecular hydrogen bonds (O3-H30…O2 and O3′-H30′…O2′) and the intermolecular hydrogen bonds (O1-H10…O3, O1′-H10′…O3′, O2-H20…O4′, and O2′-H20′…O4).

Figure 1.

A, The crystal structures of Wangzaozin A (X and X′). B, The configuration of inositol 1(or 4) monophosphatase. The blue ball structures are the reported inhibitors. The red ball structures are Wangzaozin A (X and X′). Two black balls are the Gd metals. C, Helix and strand ribbons with residue receptors. X (D1) and X′ (D2) form H bonds with residues and solvents. The highest occupied molecular orbital (HOMO) surfaces of X (E1) and X′ (E2). The electrostatic potential (ESP)-mapped density surfaces of X (F1) and X′ (F2) [Color version of the figure available online].

Chemicals and Cell Culture

Roswell Park Memorial Institute (RPMI) 1640 culture medium and trypan blue dye are from Sigma Co, fetal bovine serum is from Sijiqing Co (China), and dimethyl sulfoxide is from Sangon Co (Shanghai, Jiangsu, China). All other materials are analytical reagents. Costar cell culture plates (from Sangon Co, Shanghai, Jiangsu, China) with 6 and 24 wells are used. The utilized apparatuses also include a CO₂ incubator, an inverted microscope, and a super clean bench.

Human gastric cancer SGC-7901 cell lines are cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (v/v), penicillin (100 IU/mL), and streptomycin (100 μg/mL). The cultured procedure is maintained in a humidified incubator at 37°C and 5% CO₂.

The Antiproliferation Effect and Lethal Effect of Wangzaozin A on Tumor Cells are Assessed by Trypan Blue Exclusion

The 8 × 10⁴ cells/mL cell suspension is inoculated (0.5 mL/well) into the 24-well culture plates and cultured in the cell incubator at 37°C and 5% CO₂. The concentration values of Wangzaozin A solution are, respectively, 0, 1, 4, 8, 12, 16, and 20 µmol/L. Timing begins once the Wangzaozin A solution is added into the cell suspension. The 0.1 mL cell suspension is stained by 0.05 mL trypan blue solution for 3 to 5 minutes every 12 hours. The cell suspension and the trypan blue solution are mixed thoroughly in each staining. The live cells are counted by a blood counting chamber. After 3 repeated experiments, the cell growth curve is depicted as the averaged cell numbers (×10⁴ cells) versus the time (hours).

Cytomorphology

The treated and untreated cells are collected by centrifugation at 800 r/min and washed with cold phosphate buffer solution (PBS) twice. Then, the cells are fixed in 4% paraformaldehyde solution at 4°C for 15 minutes, washed with PBS again, and smeared on a slide. 5 μg/mL hoechst33258 is used to stain the cells, and a fluorescence microscopy (Olympus, Japan) is used to observe.

Cell Cycle Measurements

The HL-60 (human leukemia-60) cells treated by 2.75 μmol/L Wangzaozin A solution are harvested and washed twice with PBS. Then, the cells are fixed with 75% (v/v) ethanol at 4°C for at least 24 hours. At the beginning of the measurement, the cells are harvested, washed twice with PBS again, and then stained with the propidium iodide solution (50 μg/mL) supplemented by 0.1% triton X-100 and 50 μg/mL ribonuclease for 30 minutes in the absence of light at room temperature. Samples are analyzed by flow cytometer.

Molecular Dynamics Calculation

Molecular dynamics (MD), which is widely applied to study the biological macromolecule, ¹² is very valuable for understanding protein dynamic behaviors at different timescales, from fast internal motions to slow conformational changes, or even to protein folding processes. ¹³ Molecular dynamics contributes to the study on the effect of explicit solvent molecules on biomolecular system based on protein structure and stability to obtain the time-averaged properties of the system, such as density, conductivity, dipolar moment, as well as different thermodynamic parameters including interactive energies and entropies. More realistic systems, which include explicit water molecules, counterions, and even a complete membrane-like environment, can be simulated by MD. Furthermore, new properties of the system can be studied as they evolve in real time based on MD. The progress in system representation has been accompanied by methodological improvements: better force fields ¹⁴ have improved the treatment of long-range electrostatic interactions and system boundary conditions, and better algorithms have been used to control temperature and pressure. Commonly used programs include Gromacs, ¹⁵ Amber, ¹⁶ Namd, ¹⁷ and so on. Gromacs is used in this study with the default setting. Before calculation, cyclin-dependent kinase 2 (Cdk2; protein data bank [PDB] ID: 2C6O) is preprocessed with Chimera tools (http://www.cgl.ucsf.edu/mailman/confirm/chimera-users/ea0691d4f6e694173207320e56f5779d8edbf5e1). The system comprises 2955 protein atoms and 17 826 water molecules, and the calculation is run for 1 nanosecond.

Dock

Aiming at drug design, docking technique can find a correct conformation of a ligand and its receptor. ^18,19 The process of binding a small molecule to its protein target is not simple. Entropic and enthalpic factors influence the interactions between them. Some aspects further complicate the quantitative description of the process, such as the mobility of both ligand and receptor, the effect of protein environment on the charge distribution over the ligand ²⁰ and their interactions with the surrounding water molecules. To obtain the correct conformation, docking technique generates a comprehensive set of the ligand/receptor conformations and ranks them according to their calculated stabilities. Commonly used docking programs are Dock, ^21,22 AutoDock, ²³ Gold, ²⁴ and so on. It has been found that Dock shows an unexpectedly better screening performance in the enrichment rates, although each of these programs has a merit over others. ²⁵

Therefore, Dock (https://dock.compbio.ucsf.edu/retrieve_dock_distribution.html) is used in this study. Chimera tools are used to prepare the ligand (Wangzaozin A) and receptor structures for docking. Hydrogen atom addition and Gasteiger charges ²⁶ are assigned to the ligand. AMBER score implements the AM1-BCC (semi-empirical (AM1) with bond charge correction (BCC)) method with Antechamber ²⁷ for the receptor atoms and the general AMBER force field ²⁸ for the ligand. The interaction energy of ligand and receptor is calculated by using electrostatic and van der Waals energy terms. The solvation energy is calculated by a generalized born solvation model. The surface area term is derived by a fast linear combination of pairwise overlaps algorithm. ²⁹ AMBER score is calculated as:

$\mathrm{E}\left(\mathrm{C}\mathrm{o}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}\mathrm{x}\right)-\left[\mathrm{E}\left(\mathrm{R}\mathrm{e}\mathrm{c}\mathrm{e}\mathrm{p}\mathrm{t}\mathrm{o}\mathrm{r}\right)\hspace{1em}\mathrm{+}\hspace{1em}\mathrm{E}\left(\mathrm{L}\mathrm{i}\mathrm{g}\mathrm{a}\mathrm{n}\mathrm{d}\right)\right]\mathrm{,}$

where E(Complex), E(Receptor), and E(Ligand), respectively, represent the energy of the complex, receptor, and ligand. In the AMBER score calculation, the input coordinates and parameters of complex, ligand, and receptor are, respectively, read into the system of the calculation. After the system reads the input coordinates, a minimization by conjugate gradient method is performed to remove bad contacts. After MD calculation (Langevin dynamics at constant temperature), a short minimization is used to obtain the final energy of ligand and receptor system. The translation, orientation, and conformation of ligand with respect to receptor can be described by a set of values in a file named dock_in.

Results and Discussion

Protein Targets of Wangzaozin A

The structures of the 2 isomers of Wangzaozin A are, respectively, submitted to TarFisDock Web site ⁷ to extract their protein targets. Each isomer has 23 targets with good energy scores. The scores and docking results of the isomers and targets are, respectively, listed in Tables S1 and S2 (see supporting information). In Table S1, the computational binding mode of peptidyl dipeptidase (PDB ID: 1Y79) and X obtains the lowest −51.7533 kJ/mol energy score, while the binding mode of inositol-1(or 4)-monophosphatase (PDB ID: 1IMB) and X′ obtains −48.2103 kJ/mol energy score. Inositol-1(or 4)-monophosphatase is related to bipolar affective disorder disease (see supporting information). However, there is no exact disease related to peptidyl dipeptidase (see supporting information). The structure of human inositol monophosphatase with Gd³⁺ and d(l)-myo-inositol 1-phosphate has been determined by X-ray crystallography. ³⁰ Two substrates (Figure 1B, 2 blue ball structures) and 2 metals (Figure 1B, 2 black balls) are, respectively, bound into 2 active sites of phosphatase dimer. Furthermore, the binding modes of 2 substrates in 2 active sites are identical, ³⁰ that is, each substrate utilizes the same key residues (Asp 93, Ala 196, Glu 213, and Asp 220) to form the same number of hydrogen bonds with the enzyme. ³⁰ The docking results show that both structures of Wangzaozin A (Figure 1B, red ball structures), respectively, dock into the above active sites. In Figure 1B, the orientation of X and X′ differs by nearly 180°. O3 of X forms a 3.150-Å H bond (Table S3) with OE2 (second oxygen on an epsilon carbon) of the residue Glu70.B (segment of Figure 1C) that is directly related to the functional action of inositol monophosphatase by reacting to Gd³⁺ as a crucial residue. ³⁰ O3′ of X′ forms a 3.179-Å H bond (Table S3) with OE2 of the residue Glu70.A. The reaction can activate the active site nucleophile (a metal-bound water) ³⁰ and result in the inhibitory action of enzyme activity. The highest occupied molecular orbits (HOMOs, Figure 1E1 and E2, Arguslab4 software: http://www.arguslab.com) reveal that the strong electron-donating groups O3 and O3′ can form H bonds with Glu70. The electrostatic potential-mapped density surfaces of X and X′ (Figure 1F1 and F2) show that the active groups adjacent to O3 and O3′ contribute to the above strong H bonds and induce small configurational change of ribbons adjacent to Glu70 and Gd³⁺ that is a crucial active ion confirmed by mutagenesis experiments to affect the enzyme activity. This is an effective pathway of ion action therapy for manic-depressive illness. ³¹ From the HOMOs (Figure 1E1 and E2), it is clear that O4 and O4′ are crucial electron-donating groups. O4 and O4′, respectively, form 2.966 and 3.069 Å H bonds with the OG elements of the serine (Ser) 165.B and Ser165.A residues (Figure 1C, red). The Ser165 residue in each active site, with 2 oxygens extending into the site, is a crucially active residue, which mediates the strain 7 (Figure 1C, blue) and the helix 5 (Figure 1C, magenta). ³⁰ O4 and O4′ (Figure 1F1 and F2) may competitively inhibit the activity of this kinked structure by the inhibitory binding action. The Asp41 residue that mediates the helix 1 (Figure 1C, cyan) and the helix 2 (Figure 1C, dim gray) contributes to the ligand–receptor stability by forming a 2.871-Å (or 3.239 Å) H bond with O2 (or O2′). H bonds between the solvent molecules and the ligand (or enzyme) may simultaneously contribute to the stability of the ligand–receptor complex and to the inhibitory action of the ligand. From Table S3 (see supporting information), the percentage values of the H bonds formed by the oxygen 1 to 4 are, respectively, 18.5%, 29.6%, 22.2%, and 29.6%.

The Antiproliferation Effect and Lethal Effect of Cancer Cells Induced by Wangzaozin A are Assessed by Trypan Blue Exclusion

In the key regulators of complex apoptotic machinery, defects cause anomalous cell to grow. The regulators can allow neoplastic cells to live beyond their normal life span, accumulate genetic mutations, and sustain growth under hypoxia, and oxidative stress conditions. ³² Human gastric cancer SGC-7901 cell lines are treated by the Wangzaozin A solutions at different culturing time. The antiproliferation effect and lethal effect of Wangzaozin A to cancer cells are assessed by trypan blue exclusion (Figure 2A). At the lower 1.0 to 4.0 µmol/L concentration of the Wangzaozin A solution, Wangzaozin A generates the antiproliferation effect to cancer cells, and the effect improves with the increase in the concentration with an evident time effect. The proliferation of the cells is nearly paused when the concentration is 8.0 µmol/L. After culturing for 24 hours, only a few cells are dead. The lethal effect appears at the higher concentration (12.0-20.0 µmol/L) of the Wangzaozin A solution, and the effect also improves with the increase in the concentration with an evident time effect. Similar to the results from the inverted microscope, Wangzaozin A inhibits the growth of human gastric cancer SGC-7901 cell lines at the lower concentration (<4.0 µmol/L) and results in the lethal effect at the relative higher concentration (>8.0 µmol/L).

Figure 2.

(A) The antiproliferation effect and lethal effect of human gastric cancer SGC-7901 cell lines induced by Wangzaozin A. Hoechst33258 staining of the untreated (B) and 25 µmol/L Wangzaozin A-treated (C) cells. The treating time is 24 hours. The magnification is 400×.

Wangzaozin A Induces Apoptosis

The nuclear complexity of the treated and untreated SGC-7901 cells by the Wangzaozin A solution is observed by the hoechst33258 staining on the slides (Figure 2). The cellular size of the treated cells is somewhat less than the untreated ones with normal features, such as nuclei round, chromatin homogeneous, and uniform fluorescence distribution. The treated cells show the features of apoptosis, such as the appearance of chromatin condensation and margination (indicated with the arrows in Figure 2C). The morphologic change in the treated cells exhibits the induced apoptosis.

Cell cycle progression requires the coordinated interaction and activation of cyclins and cyclin-dependent kinases (CDKs), which are members of the Ser/Thr protein kinase super family and the cdc2/cdkx subfamily. CDK2, along with other CDK family homologues, is known to be important in regulating the entry into the cell cycle and the progression through it (Figure 3). In S-phase, cyclin A (CycA) binds to CDK2, activating the kinase, and recruiting substrates and inhibitors to the complex through a hydrophobic groove that is approximately 50 Å away from the CDK2 active site. ³³ The transcription factor E2F is a substrate for the CDK2/CycA complex and is phosphorylated as a prelude to the S-phase exit. The cellular response to CDK2/CycA inhibition, which has been reported to result in cell death in combination with CDK2/CycA inactivation, is thought to be greater in tumor versus normal tissue since E2F is commonly deregulated in tumor cells. ³⁴ Therefore, selective inhibition of the phosphorylation event blocks the S-phase exit and sensitizes cancer cells to apoptosis. The triazolo [1,5-A] pyrimidine is a Cdk2 inhibitor, ³⁵ which occupies the special active site of Cdk2 (Supplement Figure S1). In Figure 4, the distribution of cell cycle is examined at the indicated period of time. With the time increasing, 2.75 μmol/L Wangzaozin A solution induces a time-dependent increase of G1 cell population in HL-60 cells from 0 to 60 hours. At the same time, the number of S and G2 phase cells decreases because Wangzaozin A induces G1 phase cycle arrest in HL-60 cells. Therefore, the binding modes between the isomers of Wangzaozin A and CDK2 are investigated. After stabilizing the structure by MD, the active site docked by X (red stick structure) and X′ (blue stick structure) in Figure 3 is identical to Ref ³⁵ (Supplement Figure S1). In the active site, a 3.125-Å H bond links the oxygen atom of glutamine 125 and O2 of X. At the same time, a 2.646-Å H bond is formed by O3. However, X′ has no strong H bond (Supplement Figure S2).

Figure 3.

The crystal structure of cyclin-dependent kinase 2 (CDK2) after 1 nanosecond molecular dynamics calculation. The red atom structure is X and the blue one is X′ [Color version of the figure available online].

Figure 4.

A, Flow cytometric analysis of HL-60 cells treated by 2.75 μmol/L Wangzaozin A solution for the indicated period of time. B, Time course of cell-cycle distribution in the treated cells.

In summary, the theoretical protein targets of Wangzaozin A and the programmed cell death induced by Wangzaozin A are studied. The protein targets of Wangzaozin A are screened out by TarFisDock. Each isomer has 23 targets with good energy scores. By analyzing the isomer structures of Wangzaozin A, the results show that O4 and O4′ of Wangzaozin A are critical electron-donating groups. By counting the percentage values of the H bonds formed by the oxygen 1 to 4, the results show that the values of oxygen 2 and 4 are larger than that of oxygen 1 and 3. Wangzaozin A inhibits the growth of human gastric cancer SGC-7901 cell lines at the lower concentration (<4.0 µmol/L) and results in the lethal effect at the relative higher concentration (>8.0 µmol/L). The G1 phase cycle in HL-60 cells can be arrested by Wangzaozin A. Our future work will be focused on the physiological activity of Wangzaozin A and its effect on the function of biological macromolecules. It is hopeful to develop an effective drug to treat cancer based on the special structure of Wangzaozin A.