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New insights into the anti-hepatoma mechanism of Alisol G-metal ions complexes based on c-myc DNA
⁎Corresponding authors. 300091@njucm.edu.cn (Wei Gu), zengbingli@163.com (Li Zeng)
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Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Abstract
Abstract
This study investigated the anti-hepatoma molecular mechanism of Alisol G, which is an effective component of the Chinese medicine Alisma orientalis, in the presence of metal ions Cu2+ and Fe3+ based on c-myc DNA. Here, a combination of Alisol G and metal ions (Cu2+, Fe3+) to augment anti-hepatoma efficiencies of Alisol G has been identified by methyl thiazolyl tetrazolium (MTT) assay. Network pharmacology revealed that c-myc DNA was the potential target of Alisol G with respect to its anti-hepatoma effects. By performing multi-spectroscopic analyses, we showed that the interaction of Alisol G with c-myc DNA was a process of static quenching. The binding constants and thermodynamic constants indicated that a 1:1 complex was formed between Alisol G and c-myc DNA. Moreover, metal ions strengthened the interaction between Alisol G and c-myc DNA. Molecular docking and molecular dynamics simulation further unveiled that the higher binding affinity between Alisol G-Fe3+ complex and c-myc DNA as compared to Alisol G-Cu2+ complex. This probably resulted from the polarization of metal ions and the structural flexion of Alisol G. The C22-O31-H76 and C18-O32-H77 of Alisol G were key groups in the interaction with c-myc DNA. Addition of metal ion, had greatly changed the c-myc DNA-binding domain of Alisol G while didn’t affect the kinetic stability of the interaction, thus facilitating the insertion of Alisol G into c-myc DNA A-T base pair. Importantly, the DG113 of c-myc DNA was important for its binding to metal ions. Together, our findings suggested that Alisol G in combination with metal ions may be an efficient and promising option for the treatment of liver cancer.
Keywords
Alisol G
Metal ions
c-myc DNA
Multispectral methods
Molecular docking
Molecular dynamics simulation
1 Introduction
As the sixth most commonly diagnosed cancer across the globe, liver cancer mortality rate ranks fourth worldwide (Anwanwan et al., 2020; Mandal et al., 2018). With long-term use, chemotherapeutic drugs such as sorafenib have additional issues such as toxicity, poor tolerance of patients, and resistance (Kim et al., 2020). In this connection, traditional Chinese medicine has several advantages, including little side effects, high safety profile, stable curative effect, and wide drug source. Additionally, the search for effective anticancer compounds has gained increasing interest (Gao et al., 2021a; 2021b; Yu et al., 2021).
The traditional Chinese medicine Alismatis Rhizoma (zexie) is the dry tuber of Alisma orientale (Sam.) Juzep (Han et al., 2012). The reviewed literature shows that Alismatis Rhizoma exerts a significant anticancer effect. The main effective components of Alisma orientalis are alisol compounds, among which the content of Alisol G is the highest (Xu et al., 2015; Wang et al., 2020a; 2020b; 2020c). The antitumor mechanisms of alisol compounds include blocking the tumor cell proliferation cycle, inhibiting tumor cell metastasis, inducing apoptosis of cancer cells mediated by related apoptotic pathways, and reversing multidrug resistance of tumor cells (Jang and Lee, 2021; Li et al., 2020). Clinically, Alisma orientalis is combined with a variety of Chinese herbal pharmaceuticals for treating liver cancer, ovarian cancer, lung cancer, and gastric cancer. Moreover, domestic and international research has indicated that metal drugs have an esteemed role in anticancer therapy. However, research on metal-based traditional Chinese medicine compounds in the anticancer field is still at a preliminary stage, and the exact mechanism remains complicated. Therefore, further research is urgently needed (Englinger et al., 2019; Chen et al., 2017).
As the carrier of genetic information, DNA can make cells proliferate indefinitely and promote cell division. The abnormal expression of DNA in tumor cells has been regarded as one of the key factors causing cancer. Therefore, DNA is a common target of anticancer drugs (Hurley, 2002). Emerging reports indicated that drug molecules can produce physical mutation by interacting with DNA, thereby inhibiting tumor growth. Drug molecules affect gene expression and function through binding to DNA and destabilizing its structure. Metal ions such as Fe3+, Cu2+, Pt4+, Ni2+, Co2+, and Au3+ can promote the binding of drug molecules to DNA effectively, inducing DNA lesions and inhibiting repair processes of damage, which blocks DNA replication and double-stranded DNA unhelicalization, resulting in increased cytotoxicity and enhancement of the growth inhibitory effect of cancer cells (Fan et al., 2021; Wongsuwan et al., 2021).
In this study, the methyl thiazolyl tetrazolium (MTT) assay was performed to explore the anticancer activities of a combination between Alisol G and Cu2+ and Fe3+, and the hepatocarcinoma target c-myc DNA was screened by network pharmacology. The molecular mechanism was studied by variable temperature fluorescence spectroscopy, circular dichroism, molecular docking, and molecular dynamics to investigate the interaction between the anticancer target c-myc DNA and Alisol G and the influence of Cu2+ and Fe3+ on it. This study provides a concept for the design and development of new anticancer drugs derived from metal-based traditional Chinese medicine and offers the basis for its clinical applications.
2 Materials and methods
2.1 Reagents and instruments
Reagents: DMEM medium and phosphate buffer saline (PBS) (HyClone, USA); penicillin and fetal bovine serum (FBS) (Gibco, USA); MTT [3-(4,5-Dimethylthiazol-2)-yl)- 2,5-diphenyltetrazolium bromide] (Beyotime, China); Dimethyl sulfoxide (DMSO) and trypsin (Amresco, USA); Trypan Blue (Hengyuan Biological Technology, China); Paclitaxel reference substance(Sigma, USA); Hepatocarcinoma HepG2 cells and and normal liver HL7702 cells (Cell Resource Center, China); c-myc DNA (Sangon Biotech China): (ss1:5′-TGGGGAGGGTGGGGAGGGTGGGGAAGG-3′, ss2:3′ -ACCCCTC CCACCCCTCCCACCCCTTCC-5′); Alisol G standard (Sangon Biotech China); the structure is shown in Fig. 1; Ethidium bromide (EB, Macklin, China); Acetonitrile (LC, Merck, USA); Trihydroxymethyl aminoethane (Tris, BIO LINK, USA); Tris and hydrochloric acid (HCl) were used to prepare Tris-HCl buffer (0.50 mol·L-1) pH 7.4. Ferric chloride, copper chloride (Nanjing Liangwei Biological Technology Co., Ltd.); All other chemicals used were of analytical grade and experimental water for ultrapure water.
Instruments: LS55 fluorescence spectrometer (Perkin, USA); Chirascan circular dichroism spectrophotometer (Applied Photophysics, UK); FM-4P-TCSPC uulifetime fluorescence spectrofluorometer (Horiba Jobin Yvon, USA); HH-2 digital temperature water bath kettle (Guohua Instrument, China); MX-S Mixing instrument (SCILOGEX, USA); Analytical balance (Shangping Instrument; China); PHS-3C pH meter (Electric Scientific Instrument, China); CO2 cell incubator(Thermo Fisher Scientific, USA); Super clean bench (Purification Equipment, China); Microscope (Olympus, Japan); Hemocytometer (Qiujing, China); Cell culture plate (Corning, USA); Microplate reader (Tecan, Switzerland).
2.2 Preparation of sample solution
The mother liquor (100 μM) of Alisol G solution which was ready for cell viability experiments was prepared in DMSO and stored at −20 °C.
The mother liquor of Alisol G (5.05 × 10-4 mol·L-1) was prepared with acetonitrile and diluted with Tris-HCl buffer (0.50 mol·L-1, pH = 7.4) to obtain a concentration of 1.52 × 10-4 mol·L-1 Alisol G solution which stored at 4 ℃ was ready for spectroscopy experiments.
A certain amount of ferric chloride (FeCl3) and copper chloride (CuCl2) was weighed accurately, respectively. Tris-HCl buffer (0.50 mol·L-1, pH = 7.4) was used to prepare a reserve solution with a concentration of 5.05 × 10-4 mol·L-1 and then the diluted to obtain a metal ion solution with a concentration of 1.52 × 10-4 mol·L-1.
2.3 Preparation of double stranded c-myc DNA solution (Lei et al., 2020)
C-myc DNA was dissolved in Tris-HCl buffer (0.50 mol·L-1, pH = 7.4) at room temperature and stirred until homogeneous. The solution was placed in a water bath at 85 ℃ for 10 min, then annealed and cooled to room temperature (over 4 h). The ratio of the UV absorbance at 260 nm and 280 nm of the DNA solution was greater than 1.8, indicating that c-myc DNA was free from protein and did not require any further purification. The double-stranded c-myc DNA solution was obtained, then stored at −20 ℃ for later use.
2.4 Screening of metal ions (Wang et al., 2020a; 2020b; 2020c; Zhu et al., 2019)
All the computing work was done using the Accelrys’s Discovery Studio 3.0 (DS 3.0) package. The molecular docking calculations of Alisol G with six metal ions (Fe3+, Cu2+, Pt4+, Ni2+, Co2+, Au3+) were carried out by using DS 3.0 CDOCKER module, and the docking results were evaluated comprehensively, two metal ions with the strongest action of Alisol G were selected for follow-up study.
2.5 Cell culture (You et al., 2021; Gao et al., 2021a; 2021b)
Human hepatocarcinoma HepG2 cells and normal liver HL-7702 cells were cultured
in DMEM medium supplemented with 10% FBS, 100 units/mL penicillin and 100 μg/mL streptomycin in a fully humidified atmosphere with 5% CO2 at 37 ℃, respectively.
2.6 MTT assay (M et al., 2020; yao et al., 2021)
To measure the cell proliferation of HepG2 and HL-7702 cells were planted in 96-well plates at a density of 1.0 × 105/mL, respectively. After incubation for 24 h, the cells were treated with Alisol G, Alisol G + Cu2+, or Alisol G + Fe3+ and incubated for another 48 h. After incubation, the culture medium (100 μL) and MTT (10 μL) were co-cultured for 4 h at 37 ℃, then DMSO (110 μL) was added, and the absorbance was measured at 490 nm with a microplate reader after shaking for 10 min.
2.7 Anticancer target screening for Alisol G
2.7.1 Screening of potential targets of Alisol G (Shi et al., 2021; Qiao et al., 2020)
We first searched the PubChem database (http://pubchem.ncbi.nlm.nih.gov) with the Alisol G keyword to obtain the corresponding molecular structure. Next, to identify potential targets of Alisol G, we searched the potential targets on the TCMSP (https://tcmspw.com/tcmsp.php) and the Swiss Target Prediction databases (http://www.swisstargetprediction.ch/). The targets obtained from the database screening were imported into the UniProt database (https://www.uniprot.org/). The species was defined as “Homo sapiens”. The targets were corrected and converted into standard names to finalize the database of targets of Alisol G.
2.7.2 Screening of potential targets for liver cancer and identification of common targets for component-disease targets (Jiang et al., 2020)
A liver cancer target database was established by searching for liver cancer-related targets with the keyword “liver cancer” in the GeneCards database (https://www.genecards.org/). The cross targets of Alisol G and liver cancer were extracted by R language to obtain the potential targets of Alisol G for liver cancer.
2.7.3 Construction of Alisol G-action target network (Wang et al., 2020a; 2020b; 2020c)
Alisol G and targets were introduced into the Cytoscape software, and the network of Alisol G-liver cancer targets was constructed. In the network, nodes represented components and targets, and edges represented the interaction between components and targets. A visual network of active components-disease targets was constructed, and a series of topological parameters of the network were obtained through the “Network analyzer” function.
2.7.4 Construction of target interaction network diagram and screening of core targets (Xiang-Li et al., 2021)
We entered the anti-hepatoma target of Alisol G into the STRING database (http://string-db.org/cgi/input.pl), set the species to human (Homo sapiens) and the confidence score to >0.4. Then we hid unconnected nodes, kept other parameters unchanged, and performed the target interaction analysis to construct the target interaction network diagram. The results were input into the R language calculation to obtain a bar graph of the PPI core target.
2.8 Variable temperature fluorescence spectrometry (Guo et al.; 2021; chilom et al., 2021; Shahabadi et al., 2019; Shiri et al., 2020)
DNA and EB solution were mixed to obtain a mixed system of DNA with a concentration of 5.00 µmol·L-1 and EB with a concentration of 5.00 × 10-5 mol·L-1. Different volumes of Alisol G solution were successively added to determine the change of DNA fluorescence spectra. Then, different volumes of Cu2+ and Fe3+ solutions were added respectively to determine the effect of metal ions on the changes in the fluorescence spectrum. The measurement conditions were as follows: The excitation wavelength of DNA was 480 nm, the slit width was 15 × 15 nm, and the scanning range of the emission spectrum was 500–800 nm. The fluorescence spectrum changes of the EB-DNA system were determined. The thermodynamical parameters (ΔG, ΔS, ΔH) of the interaction between Alisol G and c-myc DNA were calculated by means of fluorescence experiments at three different temperatures (298 K, 318 K, 338 K). In order to eliminate the effect of internal fluorescence, the fluorescence intensity was corrected according to the following formula: where Fcor and Fobs represented the fluorescence intensity after and before correction at emission wavelength. A1 and A2 were the absorbance values of Alisol G at the excitation wavelength and emission wavelength respectively.
2.9 Circular dichroism measurements (Rana et al., 2021; Shahabadi and Razlansari, 2021; Shahabadi and Farhadi, 2021)
2.00 mL 5.00 μmol·L−1 c-myc DNA solution was transferred to 1cm quartz cuvette, and two portions of 10.00 μL Alisol G solution were added in turn. The mixture was evenly mixed and placed until the reaction completed. The change of circular dichroism of DNA was determined. Then different metal ion solutions were added in it to determine the effect of metal ions on the change of circular dichroism spectrum. The scanning range of 200 nm to 400 nm using a bandwidth of 1nm. The reference solution was 0.50 mol·L−1 Tris-HCl buffer (pH=7.4). The circular dichroism of c-myc DNA solution was determined.
2.10 Molecular docking (Xu et al., 2018; Xu et al., 2016a; 2016b; 2016c; Xu et al., 2014; Xu et al., 2020)
The crystal structure of c-myc DNA was obtained from the Protein Data Bank database (PDB ID: 1NKP) and prepared and defined as the receptor. The structure of Alisol G was constructed and optimized using the Sketching module in DS 3.0 and its energy was minimized with CHARMm force field. Alisol G, Alisol G + Cu2+, and Alisol G + Fe3+ were defined as the ligands, respectively. The docking were performed using Flexible Docking protocol in the CHARMm force field. The complex with the lowest binding energy was identified as the most stable conformation. Moreover, the interactions were obtained by the receptor-ligand interaction analysis.
2.11 Molecular dynamics simulation (Tabassum and Ahmad, 2021; C et al., 2020; Rampogu et al., 2020)
Molecular dynamics (MD) module of the standard Dynamics Cascade in DS 3.0 was used for MD calculations. The TIP3P water model was used as the solvent for the complex, and a layer of water molecules with a thickness of 7 Å was added to the solute outer space. At the same time, it is necessary to add an appropriate amount of sodium ions to the system to balance the excess negative charges, so as to ensure that the system is electrically neutral. Before the MD simulation, the energy of the system was optimized by the steepest descent method to eliminate the large energy collision between molecules, then the conjugate gradient method was used to minimize the second energy, and then the system was heated from 50 K to 300 K. After heating, 50 ps pre-equilibrium was carried out under constant temperature and pressure. Molecular dynamics simulations for 1 ns were performed to equilibrate the whole system, and then followed by long time simulation. The degree of closure between the two structural domains was monitored at the same time. After the equilibrium, the complex was simulated for 100 ns, using periodic boundary conditions. The PME method was used to deal with the long-range electrostatic interaction. The radius of the truncation value of the non-bond interaction was set to 14 Å, and the step size was set to 0.5 fs, every 2 ps to record the coordinate trajectory.
2.12 Statistical analysis
All data were presented as means ± SD ( ±s). Significance analysis was performed using unpaired student’s t tests for two groups and one-way analysis of variance analysis for more than two groups in SPSS19.0 software. P < 0.05 was considered as significant.
3 Results and discussion
3.1 Metal ion screening results
To screen two metal ions from six metal ions that stronger interacted with Alisol G, molecular docking was performed. The larger -CDOCKER Interaction Energy value, the stronger interaction of Alisol G with metal ions. CDOCKER Energy was the total energy of the ligand and the receptor. The lower of CDOCKER energy value, the more stable of the ligand-receptor complex. As displayed in Table 1, Fe3+ and Cu2+ stronger interaction with Alisol G as compared to other metal ions. Therefore, Fe3+ and Cu2+ were selected for follow-up studies (Zhu et al., 2019; Shao et al., 2020).
| Mental ions | -CDOCKER interaction energy (kcal mol−1) | CDOCKER energy (kcal mol−1) |
|---|---|---|
| Fe3+ | 26.53 | 99.50 |
| Cu2+ | 25.05 | 106.63 |
| Pt4+ | 0.32 | 122.32 |
| Ni2+ | 20.35 | 110.24 |
| Co2+ | 19.99 | 110.21 |
| Au2+ | 0.09 | 122.36 |
3.2 Alisol G-metal ions combinations inhibit human hepatoma HepG2 cell proliferation
To detect the effect of Alisol G and its combination with metal ions on HepG2 cells, MTT assay was used to evaluate the cell viability (Fig. 2). The results showed that, compared with the control group, cell viabilities of HepG2 cells in the Alisol G, Alisol G + Cu2+, Alisol G + Fe3+, and positive control (sorafenib) groups were significantly decreased in a dose-dependent manner (*P < 0.05, **P < 0.001). Notably, the order of the inhibitory of each group on the cell proliferation was: Alisol G + Fe3+ > Alisol G + Cu2+ > Alisol G, indicating that the addition of metal ions enhance the inhibitory of Alisol G on the proliferation of HepG2 cancer cells and the Alisol G-Fe3+ combination was stronger. Moreover, Alisol G displayed no cytotoxicity to normal liver HL7702 cells, suggesting that Alisol G was highly biosafe.
A. The effects of different concentrations of Alisol G and the addition of metal ions on the proliferation of HepG2 and HL7702 cells.
B. The effects of 100 μM Alisol G and the addition of metal ions on the proliferation of HepG2 and HL7702 cells; compared with the blank group.
3.3 Screening of anti-hepatocarcinoma target of Alisol G
TCMSP and the Swiss Target Prediction databases were combined to get 78 targets of Alisol G. A total of 749 liver cancer targets were retrieved and screened from the GeneCards database. 31 targets of the Alisol G-liver cancer intersection were obtained by R language. The potential targets are listed in Table 2. The potential anti-hepatoma targets of Alisol G were introduced into Cytoscape software to construct the graph of the Alisol G-liver cancer target network. As shown in Fig. 3A, there are 32 nodes with 31 edges in the network. The green rhombic node represents Alisol G, and the blue elliptical nodes represent the action targets, whereas the connecting lines show the interaction between Alisol G and the corresponding target. The results showed that Alisol G exhibited anti-hepatoma effects by acting on 31 targets.
| Compound | Targets |
|---|---|
| Alisol G | CYP19A1, CYP17A1, ESR2, AR, TERT, PRKCA, PTGS2, PPARG, ESR1, ABL1, CCND1, CDK4, CDK2, CCNE1, KDR, MAPK14, VDR, MYC, EGFR, JAK3, NR3C2, FGFR1, MDM2, MTOR, PARP1, PGR, KIT, TEK, PIK3CB, AURKA, PDGFRB |
The PPI network was obtained by inputting the common targets of Alisol G-liver cancer into the string data platform. The result is presented in Fig. 3B. The PPI network diagram contains 31 nodes and 215 edges with an average node degree value of 13.871, a clustering coefficient being 0.356, and network density being 0.356 in the graph. The nodes represent the target points, and each edge represents the interaction between the targets. More emerging lines from a node reflect a greater correlation of that target, indicating its more critical and important role. As evident from Fig. 3B, MYC, MTOR, and CCND1 are situated in the central area of the network and connect with many nodes, suggesting that they have a core position in the network. The tsv file downloaded in the string was processed by R language to calculate the core target bar graph. The higher corresponding value for a target in the core target bar graph further implies its increased correlation with other targets, indicating its significant role in the network. The core target bar graph is shown in Fig. 3C, with the highest value for MYC.
A. Alisol G-liver cancer target network diagram.
B. PPI network diagram.
C. Core target bar graph of PPI.
The diagram shows the synthesis of the Alisol G-liver cancer target network and the core target diagram of the PPI network. It can be seen that MYC has the most lines and the highest number of bars. It is the target with the largest number of adjacent nodes, which shows that it plays a key role in the network and is the core gene. The Uniprot database was used to query MYC, and its corresponding gene was c-myc DNA. Therefore, in this paper, c-myc DNA was selected as the core target of the anti-hepatocarcinoma effect of Alisol G, and the interaction between Alisol G and c-myc DNA was analyzed.
3.4 Variable temperature fluorescence spectrum experiment
3.4.1 Fluorescence quenching mechanism
Fluorescence spectroscopy is an important method for studying the interaction between drug molecules and DNA (Ahmad et al., 2016). Information of the interaction such as the binding constant and thermodynamic parameters can be obtained by using variable-temperature fluorescence and then the interaction mode can be judged. The interaction between Alisol G and c-myc DNA was studied by variable temperature fluorescence spectroscopy. Given that DNA is not fluorescent, it is necessary to form an EB-DNA fluorescent probe system with the use of fluorescent probe molecule EB. At room temperature (298 K), the fluorescence spectra of the interaction between Alisol G and c-myc DNA were shown in Fig. 4A-C. With the continuous addition of Alisol G, the fluorescence intensity of the EB-DNA system was significantly reduced, and the peak position did not shift. It was speculated that the Alisol G could compete to replace EB and interpose with c-myc DNA. Metal ions Cu2+ and Fe3+ were added to Alisol G respectively. With the addition of Cu2+ and Fe3+, the fluorescence intensity of EB-c-myc DNA was significantly reduced. It was suggested that the addition of metal ions promotes the interaction between Alisol G and c-myc DNA, which is beneficial to its insertion into c-myc DNA.
The rate of change of fluorescence intensity of Alisol G and metal ions in EB-DNA system was calculated. As shown in Table 3, the fluorescence change rate of Alisol G was 9.32%. After the addition of Cu2+, the fluorescence change rate of Alisol G was 12.78%. After adding Fe3+, the fluorescence change rate of Alisol G was 14.46%. The decreasing order was as follows: Alisol G + Fe3+ > Alisol + Cu2+ > Alisol G. The results showed that the addition of metal ions promoted the insertion of Alisol G into c-myc DNA and the effect of Fe3+ was stronger than that of Cu2+.
| Groups | Δ(%)* |
|---|---|
| c-myc DNA + Alisol G | −9.32 |
| c-myc DNA + Alisol G + Cu2+ | −12.78 |
| c-myc DNA + Alisol G + Fe3+ | −14.46 |
Note: *Δ(%)= (Fcor-Fcor,0)/Fcor ,0 × 100% (where Fcor,0 is the fluorescence absorption intensity of the DNA solution without adding the drug after correction, and Fcor is the correction after adding the drug fluorescence absorption intensity of the DNA solution).
Fluorescence spectroscopy experiments of the interaction between Alisol G and DNA at 318 K and 338 K were carried out. The fluorescence spectra of Alisol G at 298 K, 318 K and 338 K at three different temperatures were measured according to the Stern-Volmer equation Fcor, 0 / Fcor = 1 + Kqτ0 [Q] = 1 + KSV [Q], (Fcor, 0 and Fcor are the fluorescence intensity before and after the addition of the corrected quencher, τ0 is the average lifetime of fluorescence molecule, [Q] is the quencher concentration), as shown in Fig. 4D. The quenching curve is linear, indicating that there is only one quenching mechanism between them. The quenching constant KSV and quenching rate constant Kq of the interaction was calculated according to the curve.
The double logarithmic regression curve was used to calculate the binding constant (Ka) and the number of binding sites (n) of the interaction between the two.
The binding constant and the number of binding sites of the two interactions at different temperatures were calculated according to the slope and intercept of to . The calculation results are listed in Table 4. The results are listed in Table 4, the value of the quenching rate constant (Kq) is much larger than the maximum collision rate constant (2 × 1010 L·mol−1·s−1) of the dynamic quenching process, and the value of the quenching constant (KSV) decreases with the increase of temperature, and Ka decreased with the increase of temperature, the results showed that static quenching was the only quenching mechanism of the interaction between Alisol G and c-myc DNA. n was approximately 1, indicating that Alisol G combined with c-myc DNA and formed a 1:1 complex.
| T/K | Ksv / ×104 L mol−1 | Kq / ×1012 L mol−1 s−1 | Ka / L mol−1 | N |
|---|---|---|---|---|
| 298 K | 5.29 | 5.29 | 1.99 × 108 | 1.28 |
| 318 K | 4.91 | 4.91 | 7.04 × 103 | 0.86 |
| 338 K | 2.41 | 2.41 | 2.20 × 103 | 0.78 |
3.4.2 Results of thermodynamic parameters
The thermodynamic parameters of the main interaction types were analyzed by using the van’t Hoff equation.
In the formula, R is a thermodynamic constant, and its value is 8.314 J·mol−1·K-1, T is the thermodynamic temperature (298 K, 318 K, 338 K), ΔH is the enthalpy change of the system after interaction, ΔS is the entropy change of the system after interaction, and ΔG is the change of binding energy of the system after interaction, the value of ΔH is the change of enthalpy of the system after action, ΔS is the change of entropy of the system after action, and ΔG is the change of binding energy of the system. As shown in Fig. 4E, ΔH and ΔS values can be calculated according to the slope and intercept of the curve, ΔH=-236.40 KJ·mol−1, ΔS=-633.81 J·mol−1·K−1. The values of ΔH and ΔS are substituted into the Gibbs-Helmholtz equation ΔG=ΔH-TΔS to calculate ΔG, ΔG(298K)=-47.52 KJ·mol−1, ΔG(318K) = -34.58 KJ·mol−1, ΔG(338K)=–22.17 KJ·mol−1. ΔG<0, ΔH<0 and ΔS<0 suggested that the binding of Alisol G to c-myc DNA was a spontaneous exothermic process. Hydrogen bond and van der Waals force are the main forces of Alisol G and c-myc DNA.
A. Fluorescence spectrum of Alisol G with the addition of c-myc DNA solution (298 K) (a: c-myc DNA; b:c-myc DNA + 5.00 μL Alisol G; c: c-myc DNA + 10.00 μL Alisol G; d: c-myc DNA + 15.00 μL Alisol G; e: c-myc DNA + 20.00 μL Alisol G; f: c-myc DNA + 25.00 μL Alisol G).
B. After adding Cu2+, the fluorescence spectrum of Alisol G with the addition of c-myc DNA solution (298 K) (a: c-myc DNA; b: c-myc DNA + Alisol G; c: c-myc DNA + 5.00 μL Alisol G + Cu2+; d: c-myc DNA + 10.00 μL Alisol G + Cu2+; e: c-myc DNA + 15.00 μL Alisol G + Cu2+; f: c-myc DNA + 20.00 μL Alisol G + Cu2+; g: c-myc DNA + 25.00 μL Alisol G + Cu2+).
C. After adding Fe3+, the fluorescence spectrum of Alisol G with the addition of c-myc DNA solution (298 K) (a: c-myc DNA; b: c-myc DNA + Alisol G; c: c-myc DNA + 5.00 μL Alisol G + Fe3+; d: c-myc DNA + 10.00 μL Alisol G + Fe3+; e: c-myc DNA + 15.00 μL Alisol G + Fe3+; f: c-myc DNA + 20.00 μL Alisol G + Fe3+; g: c-myc DNA + 25.00 μL Alisol G + Fe3+).
D. Sterm-Volmer curve of temperature-changing fluorescence experiment of Alisol G and c-myc DNA solution.
E. The van’t Hoff curve of the interaction between Alisol G and c-myc DNA (298 K, 318 K, 338 K).
3.5 Circular-dichroism study
To understand the conformation behavior of c-myc DNA and binding with small molecules has been studied by circular dichroism (CD) spectra. As shown in Fig. 5, the CD spectrum of c-myc DNA was characterized by a positive peak at 270 nm and a negative peak at 240 nm. The presence of the positive peak and negative peak had been attributed to the base stacking and DNA double helix, respectively. Moreover, the titration of c-myc DNA with both the ligand Alisol G and Alisol G-metal ions complexes lead to an increase in the intensity of positive and negative peaks followed unchanged peak position. Hence, it demonstrated that Alisol G formed an intercalation with c-myc DNA and the addition of metal ions enhanced their interaction.
In addition, the rates of change in the CD intensity c-myc DNA with both the ligand Alisol G and Alisol G-metal ions complexes were calculated (Table 5). The order of the interaction intensity was Alisol G + Fe3+ (10.91%) > Alisol G + Cu2+ (9.87%) > Alisol G (6.07%), which was consistent with the results of MTT assay and fluorescence spectrum. Compared with the addition of Alisol G, the CD peak intensity increased after the addition of Alisol G-metal ions complexes, indicating that the addition of metal ions promoted the insertion of Alisol G into c-myc DNA and enhanced their interaction. Compared with the Alisol G-Cu2+ complex, the Alisol G-Fe3+ complex stronger interacted with c-myc DNA.
| Groups | Δ(%)* |
|---|---|
| c-myc DNA + Alisol G | 6.07 |
| c-myc DNA + Alisol G + Cu2+ | 9.87 |
| c-myc DNA + Alisol G + Fe3+ | 10.91 |
A. c-myc DNA.
B. Alisol G + c-myc DNA (a: c-myc DNA; b: c-myc DNA + 10.00 μL Alisol G; b: c-myc DNA + 20.00 μL Alisol G).
C. Cu2++Alisol G + c-myc DNA (a: c-myc DNA; b: c-myc DNA + 10.00 μL Alisol G + Cu2+; c: c-myc DNA + 20.00 μL Alisol G + Cu2+).
D. Fe3++Alisol G + c-myc DNA (a: c-myc DNA; b: c-myc DNA + 10.00 μL Alisol G + Fe3+; c: c-myc DNA + 20.00 μL Alisol G + Fe3+).
3.6 Molecular docking results
Molecular docking is a theoretical simulation method which uses computer technology to calculate the binding model between ligand and receptor based on the principle of molecular geometric matching and energy matching (Ahmad and Ahmad, 2018; Arif et al., 2020). The mode of interaction between Alisol G and c-myc DNA was further studied by molecular docking. C-myc DNA is a B-type DNA fragment with four hydrophobic bases on the inside of the helix and negatively charged hydrophilic phosphate and deoxyribose units on the outside. The recognition sites of DNA targeting molecules are composed of parallel stacked bases, polymerized anionic phosphate skeleton and large and small grooves formed by two deoxynucleotides.
The molecule of Alisol G was defined as a ligand and docked with c-myc DNA. In addition, metal ions Cu2+ and Fe3+ were added to the system to study the influence of metal ions on the interaction between Alisol G and c-myc DNA. Considering the interaction parameters of the ligand-receptor complex and the formation of hydrogen bond between them were taken into account to judge the maximum binding affinity and the optimal conformation of the ligand-c-myc DNA complex.
3.6.1 Molecular docking results of Alisol G and c-myc DNA
The fluorescence spectra showed that Alisol G combined with c-myc DNA to form a 1:1 complex. Therefore, a molecule of Alisol G was chosen to docking with a molecule of c-myc DNA. The results were shown in Fig. 6. Fig. 6A was the overall picture of the interaction between Alisol G and c-myc DNA, c-myc DNA is composed of f chain and g chain. The parent ring of Alisol G is inserted into the DT112-DA309 and DG113-DC308 base pairs of c-myc DNA double helix. Fig. 6B showed the interface diagram of the interaction between Alisol G and c-myc DNA. As shown in the figure, part of the side chain of Alisol G was bound to the groove of DNA. Fig. 6C depicted the hydrogen bond diagram between Alisol G and c-myc DNA. The C18-O32-H77 of Alisol G formed hydrogen bond with the OP2 of c-myc DNA f-chain thymidine deoxynucleotides (DT112). The specific information was presented in Table 6.
| Alisols | X—H…Y | d（X—H) | d(H…Y) | d(X…Y) | ∠XHY |
|---|---|---|---|---|---|
| Alisol G | C18:O32-H77…OP2(f chain: DT112) | 0.95 | 2.16 | 2.82 | 38.58 |
The change of conformation of Alisol G before and after docking was shown in Fig. 6D-F. Fig. 6D showed that the structure of the small molecule changes obviously. Fig. 6E depicted that the angle of the side chain of small molecules had changed from 121.15° to 119.24°, and C19-C20-C17 from 122.36° to 112.82° after docking. Fig. 6F showed that the dihedral angle C18-C19-C20-C17 had changed from 58.91° to −68.46°. The changes of small molecule angle and dihedral angle demonstated that the stretching degree of small molecule structure increases after docking. Fig. 6G showed the projection of the Alisol G + c-myc DNA complex. As shown in the figure, the radius of curvature of the small molecule was 17.92 Å, which was close to the curvature of 13.75 Å of c-myc DNA, indicating that the small molecule can be inserted into the base pair of c-myc DNA.
The root mean square deviation (RMSD) of Alisol G was calculated to be 1.30 Å. RMSD is used to measure the movement amplitude of each atom in a molecule in molecular docking. The larger value indicated that a more drastic change in the position of each atom in the molecule and a greater conformational change. Before docking, the surface area of small molecules was 541.03 Å and after docking was 561.07 Å, which increased by 3.70%. The electrostatic energy of the interaction between Alisol G and c-myc DNA was −10.46 kcal·mol−1, and the van der Waals energy was −13.20 kcal·mol−1. The van der Waals energy between the systems was lower than the electrostatic energy, indicating that the binding of small molecules to c-myc DNA was dominated by van der Waals force. The calculated binding energy ΔG° between Alisol G and c-myc DNA was −45.61 kcal·mol−1, which was consistent with the experimental results of variable temperature fluorescence spectroscopy, indicating that the molecular docking results of this system were credible.
3.6.2 The molecular docking results of Cu2++ Alisol G + c-myc DNA
The molecular docking results of the interaction between Alisol G and c-myc DNA after addition of Cu2+ were shown in Fig. 7. Fig. 7A showed the overall diagram of the interaction between Alisol G and c-myc DNA. As shown in the figure, the parent ring structure of Alisol G was embedded into the base pairs of the double helix skeleton of c-myc DNA. The active region included three base pairs of DG113-DC308, DC114-DG307, DT115-DA306. Fig. 7B depicted the interface diagram of the interaction between Alisol G and c-myc DNA, which indicated that the side chain of Alisol G inserted into the groove region of DNA. Fig. 7C showed the action of Cu2+. As shown in the figure, Cu2+ acted on the parent ring region of Alisol G.
In Fig. 7D-F, the conformational changes of Alisol G after docking was prensented. Fig. 7D showed that the small molecule conformation changes significantly after docking. Fig. 7E showed that the angle of C18-C19-C20-C17 became 113.77°, the angle of C19-C20-C17 became 111.35°, and the dihedral angle of C18-C19-C20-C17 became −147.93°. The changes of series angles manifested that the structural extension of small molecules after docking was stronger than monomers.
Fig. 7G showed the distance between Cu2+ and Alisol G. As shown in the figure, the distance between Cu2+ and the carbonyl C2-O30 in the parent ring of Alisol G was the closest, which was 3.72 Å. Fig. 7H depicted the potential interface of the interaction between Cu2+ and Alisol G. As shown in the figure, the binding region C2-O30 of Alisol G was red, indicating that the electronegativity was negative while Cu2+ had a positive charge and produced electrostatic attraction, which was beneficial to the binding of small molecules to DNA. Fig. 7I showed the projection of Cu2++ Alisol G + c-myc DNA complex. As depicted in the figure, the radius of curvature of small molecules was 17.38 Å.
The RMSD of Alisol G was calculated to be 6.93 Å under the action of Cu2+. After docking, the surface area of small molecules became 567.07 Å, which increased by 4.81%. The electrostatic energy between Alisol G and c-myc DNA was −10.81 kcal·mol−1, and the van der Waals energy was −9.11 kcal·mol−1. The electrostatic energy was lower than van der Waals energy between Alisol G and c-myc DNA, which indicated that the interaction between small molecule and c-myc DNA was mainly electrostatic. The binding energy ΔG° between Alisol G and c-myc DNA was calculated to be −72.34 kcal·mol−1.
The calculation results showed that after the action of Cu2+, the value of RMSD, increase of surface area, the torsion of side chain, proximity of radius of curvature to DNA of Alisol G were stronger than the monomer, and the ΔG° value was lower than the monomer as well, indicating that Cu2+ was beneficial to the intercalation of small molecules with c-myc DNA.
3.6.3 The molecular docking results of Fe3+ and Alisol G with c-myc DNA
Fig. 8 described that the molecular docking results of the interaction of Fe3+ and Alisol G with c-myc DNA. Fig. 8A was the overall diagram of the interaction between Alisol G and c-myc DNA. The parent ring structure of Alisol G was embedded in the base pairs of the double-helix skeleton of c-myc DNA, and its active pocket contained the four base pairs of DG113-DC308, DC114-DA307, DT115-DA306 and DA116-DT305. Fig. 8B showed the interface diagram of the interaction between Alisol G and c-myc DNA. The side chain of Alisol G bound to the DNA groove. Fig. 8C was the local map of Fe3+ action, indicating that Fe3+ acted on the side chain region of Alisol G. Fig. 8D was the hydrogen bond diagram between Alisol G and c-myc DNA. The C22-O31-H76 of Alisol G formed hydrogen bond with the OP2 of c-myc DNA f-chain thymidine deoxynucleotides (DT112). The hydrogen bond information was shown in Table 7.
| Alisols | X—H…Y | d（X—H) | d(H…Y) | d(X…Y) | ∠XHY |
|---|---|---|---|---|---|
| Alisol G | C22:O31-H76…OP2(f-chain: DT112) | 0.95 | 2.41 | 2.69 | 62.98 |
The structural change of Alisol G after docking was shown in Fig. 8E-G. Fig. 8E showed that the conformation of Alisol G changed obviously after docking. As shown in the Fig. 8F, the angle of side chain of small molecule ∠C18-C19-C20 turned into 112.76° and ∠C19-C20-C17 turned into 112.48°. As shown in the Fig. 8G, the dihedral angle C18-C19-C20-C17 was −166.76°. The change of series angle indicated that the extensibility of small molecular structure increased further after the addition of Fe3+.
Fig. 8H showed the distance between Fe3+ and Alisol G. As shown in the figure, the distance between Fe3+ and the hydroxyl O31-H76 in the side chain of Alisol G is 2.43 Å. Fig. 8I was the potential interface diagram of the interaction between Fe3+ and Alisol G. The binding region C22-O31-H76 of Alisol G was red, indicating that its electronegativity was negative, while Fe3+ had three positive charges, which produced electrostatic attraction and promoted the binding of small molecules with DNA. Fig. 8J showed the projection of Fe3++Alisol G + c-myc DNA complex. As shown in the figure, the radius of curvature of small molecules was 16.32 Å.
It is calculated that after the addition of Fe3+, the RMSD of Alisol G was 9.15 Å. After docking, the surface area of small molecules becomed 572.85 Å, which was 5.88% higher than that before docking. The calculated electrostatic energy of the interaction was −9.92 kcal·mol−1, and the van der Waals energy was −8.03 kcal·mol−1. The electrostatic energy between the system was lower than that of van der Waals energy, indicating that there was mainly electrostatic interaction between small molecules and c-myc DNA. The binding energy ΔG° between Alisol G and c-myc DNA was calculated to be −78.31 kcal·mol−1.
The results showed that the conformational change, surface area increase, the torsion of side chain and proximity of radius of curvature to DNA of Alisol G after adding Fe3+ were stronger than adding Cu2+, and the binding energy of Fe3+ was lower than the addiotion of Cu2+. It showed that the insertion of Fe3+ also enhanced the intercalation of Alisol G and c-myc DNA, and the interaction intensity was higher than that of Cu2 +.
The results of molecular docking showed that the binding strength of Alisol G and metal ions to c-myc DNA was in the order of Alisol G + Fe3+ > Alisol G + Cu2+ > Alisol G. The difference in binding strength was caused by two factors:
(1) Active region differences in DNA: Alisol G bound to the DT112-DA309 and DG113-DC308 base regions of c-myc DNA, and its binding active region contained two base pairs. The active regions in Cu2+ binding to Alisol G were DG113-DC308, DC114-DG307, and DT115-DA306, containing three base pairs. Fe3+ bound to Alisol G in the DG113-DC308, DC114-DA307, DT115-DA306, and DA116-DT305 base regions, which contained four base pairs. The order of the active region size was Fe3+ > Cu2+ > monomer, which was consistent with the binding intensity of Alisol G to c-myc DNA, indicating that an increase in the active region was beneficial to the interaction.
(2) Structural differences in small molecules: A. Surface area: compared to before docking, the surface area of Alisol G increased by 5.88% after the addition of Fe3+ and increased by 4.81% after adding Cu2+. The increase in surface area by Fe3+ was greater than that of Cu2+, which indicated the combination range expanded after the addition of Fe3+. B. Spreading degree of the structure: after adding Fe3+, the dihedral angle of the small molecule changed from 58.91° to −166.76°, and after adding Cu2+, the dihedral angle changed to −147.93°. After the addition of Fe3+, the spreading of the small molecule structure was greater than that from the addition of Cu2+, which was beneficial to the intercalation of Alisol G into c-myc DNA and the binding from the addition of Fe3+ was stronger than that of Cu2+. C. Binding distance: the nearest group between Fe3+ and the small molecule was the hydroxyl C22-O31-H76 in the side chain, which was 2.43 Å away, and the closest group between Cu2+ and the small molecule was carbonyl C2-O30 in the parent ring, which was 3.72 Å away. The closer the molecular distance, the stronger the interaction, so the binding strength of Fe3+ was stronger than that of Cu2+. At the same time, it can be inferred that the highly charged metal ions were more prone to interacting with hydroxyl in the side chain of the small molecules, whereas the lesser charged metal ions were more prone to interacting with carbonyl in the parent ring. D. Degree of polarization: the charge number of Fe3+ was +3, the charge number of Cu2+ was +2, the ion radius of Fe3+ was 60 pm, the radius of ion of Cu2+ was 73 pm. The larger the charge number indicated that the radius become smaller and the degree of ion polarization turn into stronger. Therefore, the degree of polarization of Fe3+ ions was stronger than that of Cu2+. As a result, the DNA electron cloud in the active region was more deformed, and it was easier for the positive and negative electron clouds to overlap, which made the binding stronger than that of Cu2+. E. Hydrogen bonds: after the addition of metal ions, the interaction between Alisol G and c-myc DNA changed from van der Waals forces to electrostatic forces, and a hydrogen bond was formed after the addition of Fe3+, but no hydrogen bond was formed after the addition of Cu2+. The increase in hydrogen bonds suggested that the polarization effect of the small molecule system become stronger. And then the electrostatic interaction of the small molecule system become stronger. Thus, the binding of Fe3+ was stronger than that of Cu2+.
3.7 Molecular dynamics results
3.7.1 Molecular dynamics results of Alisol G and c-myc DNA
The interaction mechanism of Alisol G and c-myc DNA was studied by 100 ns (ns) molecular dynamics simulation. The results were shown in Fig. 9. Fig. 9A was a general diagram of the interaction between Alisol G and c-myc DNA. A rectangular water box with a radius of 7 Å was added around c-myc DNA and Alisol G was surrounded by a small blue threshold. Fig. 9B was the twist angle of different bases of c-myc DNA. When c-myc DNA bound to Alisol G, the bases were distorted to different degrees. The twist angle of the double helix base of Alisol G and c-myc DNA complex was calculated and the result was shown in Fig. 9B. Compared with A-T base pair, the twist angle of G-C base pair was more obvious. The binding of small molecules to c-myc DNA leads to a large twist of the G-C base pair of DNA double helix, indicating that Alisol G was intercalated with G-C base. Fig. 9C showed the radius of gyration (Rg), radius of gyration of c-myc DNA after interaction can be used to measure the compactness of macromolecular structure. The smaller the radius of gyration value, the greater the compactness, and the larger the structure, the more expanded. The analysis from the figure showed that when Alisol G and c-myc DNA begin to combine, the spatial structure of c-myc DNA changed and the system expanded, and then over time, the Rg curve tends to be smooth and the fluctuation decreases, indicating that the complex forms a stable structure. Fig. 9D showed the change of RMSD of the complex of Alisol G and c-myc DNA with time. RMSD is an important parameter used to measure the volatility of the system in molecular dynamics simulation. The greater the RMSD value, the greater the degree of denaturation. When the RMSD value tended to be constant and fluctuates slightly in a certain range, the system tends to be stable. The analysis in Fig. 9D showed that the RMSD of the system was rising rapidly at the beginning of the simulation, indicating that Alisol G interacts with c-myc DNA and the structure changes. After 20 ns, the fluctuation of RMSD became smaller, and the system tended to be stable, indicating that the structure of the complex was stable, which also corresponded to the result diagram of the radius of gyration. Fig. 9E showed the interaction energy curve of the two, and its van der Waals energy was lower than the electrostatic energy, indicating that the interaction between small molecules and c-myc DNA was dominated by van der Waals force, which was consistent with the results of molecular docking, indicating that the molecular docking results had good kinetic stability.
3.7.2 The molecular dynamics results of Cu2++ Alisol G and c-myc DNA
The molecular dynamics results of interaction between Cu2+ + Alisol G and c-myc DNA were shown in the Fig. 10. Fig. 10A was the molecular dynamics structure of Cu2+ and Alisol G and c-myc DNA. C-myc DNA was surrounded by a rectangular water box with a radius of 7 Å. Fig. 10B showed the local diagram of the interaction between Alisol G and c-myc DNA after adding Cu2+, and Alisol G and Cu2+ were surrounded by a small blue sill. Fig. 10C displayed the change of the twist angle of different base pairs of c-myc DNA after the addition of Cu2+, and the change of the torsion angle of A-T base pair was significantly larger than that of G-C base pair, indicating that the interaction between Cu2+ +Alisol G and the A-T base pair of c-myc DNA was more obvious. It manifested that the binding site of Alisol G and c-myc DNA was changed from G-C base pair to A-T base pair after addition of Cu2+. Fig. 10D showed the distance from Cu2+ to the bases within the binding range of c-myc DNA. As shown in the figure, the distance from Cu2+ to DG113 bases varied the most, so it showed that DG113 may be the binding site of Cu2+ in c-myc DNA. Fig. 10E was the radius of gyration graph of Cu2++ Alisol G + c-myc DNA, which showed that the complex radius of gyration decreased and the system structure shrunk after 20 ns simulation, indicating that the structure of the Cu2++ Alisol G + c-myc DNA complex was stable. Fig. 10F was the RMSD diagram of the system, which showed that the structure of the composite was stable after 20 ns dynamics simulation. Fig. 10G was the curve of interaction energy of Cu2++ Alisol G + c-myc DNA. As shown in the figure, the electrostatic energy of the system was lower than that of van der Waals, indicating that the interaction between Cu2+ and Alisol G and c-myc DNA was mainly electrostatic interaction. The average binding energy of Cu2+ and Alisol G was lower than that of Alisol G monomer, indicating that the interaction between Alisol G and c-myc DNA was stronger than that of monomer, which was consistent with the results of molecular docking, indicating that the system had kinetic stability.
3.7.3 The molecular dynamics results of Fe3++ Alisol G and c-myc DNA
The molecular dynamics results of Fe3+ and Alisol G and c-myc DNA were shown in Fig. 11. Fig. 11A showed the molecular dynamics structure of Fe3+ and Alisol G and c-myc DNA. A rectangular water box with a radius of 7 Å was added around the system. Fig. 11B showed the local diagram of the interaction between Alisol G and c-myc DNA after adding Fe3+. Alisol G and Fe3+ were surrounded by a small blue sill. Fig. 11C depicted that the change of the twist angle of different bases of c-myc DNA after adding the interaction of Fe3+, and the change of the twist angle of A-T base pair was greater than that of G-C base pair which showing that the binding site of Alisol G and c-myc DNA was changed from G-C base pair to A-T base pair after the addition of Fe3+. Fig. 11D showed the distance from Fe3+ to the bases within the binding range of c-myc DNA. As shown in the figure, the distance from Fe3+ to DG113 bases varied the most, and it demonstrated that DG113 was the binding site of Fe3+ and c-myc DNA. Fig. 11E the radius of gyration diagram displayed that after adding Fe3+, the Rg curve tended to be smooth and the complex system was stable after 20 ns, which depicted that the addition of Fe3 + did not affect the structural stability of Alisol G + c-myc DNA. Fig. 11F was the RMSD diagram of the complex, which showed that the system was gradually stable after 20 ns kinetic simulation. Fig. 11G was the interaction energy curve. The electrostatic energy of the system was lower than that of van der Waals, indicating that the interaction of Fe3+ and small molecules with c-myc DNA was mainly electrostatic interaction, and its average binding energy was lower than that of Cu2+ docking with Alisol G and c-myc DNA. It showed that the binding ability of Fe3+ was stronger than that of Cu2+, which was consistent with the results of molecular docking, which further indicates that the molecular docking result of this system was reliable.
The molecular dynamics results showed that Alisol G bound to the G-C base pair of c-myc DNA. After the addition of metal ions, Alisol G bound to the A-T base pair of c-myc DNA, and the addition of metal ions promoted the binding of Alisol G to c-myc DNA. Changes in the RMSD value, Rg, and the average binding energy were consistent with the molecular docking results, indicating that the dynamics of the system were stable. The binding strength order of Alisol G + Fe3+ > Alisol + Cu2+ > Alisol G was not the result of an instantaneous molecular collision, but a continuous and normal stable behavior. The binding site of Cu2+ and Fe3+ in c-myc DNA was DG113.
3.8 Discussion
To date, surgical treatment is the only possible treatment for patients with liver cancer. However, sorafenib, the most suitable drug, is also used to treat liver cancer, which can improve the quality of life of patients and extend overall survival. However, most patients will develop resistance to sorafenib, and the combination regimen is affected by adverse reactions and patient's physical condition. Therefore, alternative strategies for the treatment of liver cancer are required immediately (Yang et al., 2021; Wen et al., 2021). Traditional Chinese medicine emerges as an appealing option with high safety profile due to negligible side effects, stable curative effects, and extensive drug sources. Thus, an inclination in research to find new antitumor compounds from traditional Chinese medicine is observed (Wang et al., 2021).
Alisol acetates are the main active ingredients of the Alisma orientalis, with substantial potential in enhancing the efficacy of cancer chemotherapy and cancer treatment due to their anticancer effects through mediating relevant apoptotic pathways, reversing multidrug resistance and inducing autophagy in tumor cells (Jang and Lee, 2021). In alisol acetates, the monomer Alisol G is present in higher amounts (Liu et al., 2020a; 2020b).
Domestic and international studies have demonstrated that metal-based drugs have tremendous potential in developing anticancer drugs. In recent decades, the understanding of the anticancer mechanism of such drugs has increased; however, the research is still in its infancy. The reason for sluggish developments is that the studied compounds are poorly selective in cancer cells, and the mechanism of action remains vague (Liu and Ran, 2020). Therefore, strategic research in this field is a pressing necessity (Falsafi et al., 2021).
Tumorigenesis usually results from abnormal expression of many genes, specifically the overexpression of oncogenes and inactivation of tumor-suppressor genes. C-myc DNA is one of the principal proto-oncogene in human beings, and its overexpression can make cancer cells proliferate indefinitely and promote their division. It is closely related to the growth of many tumors (Liu et al., 2020a; 2020b; Aggarwal and Bhavesh, 2021).
In this regard, the anticancer effect and molecular mechanism of Alisol G before and after the addition of metal ions based on c-myc DNA are studied in this paper. To the best of our knowledge, no such studies have been reported in the literature.
In this study, a gene target c-myc DNA for the treatment of liver cancer was screened by network pharmacology and detected by using a combination of the MTT method, variable temperature fluorescence spectrometry, circular dichroism, molecular docking, and molecular dynamics. The results showed that the addition of metal ions could effectively promote its binding to c-myc DNA, thus preventing c-myc DNA replication and double-stranded DNA unhelicalization, enhancing its inhibitory effect on the proliferation of hepatocellular carcinoma cells. The Fe3 + showed the strongest above described promoting effects.
Further analysis of the differences in the binding strength of Alisol G towards c-myc DNA upon adding metal ions from a molecular structure perspective by molecular docking showed that two factors led to a higher binding strength of Alisol G + Fe3+ compared to Alisol G + Cu2+ and Alisol G alone- (i) Active region differences in DNA: The base-pair binding of Alisol G to c-myc DNA is less than Cu2+ + Alisol G, whereas it was highest for the combination of Fe3+ + Alisol G. This is consistent with the binding intensity of Alisol G to c-myc DNA, indicating that increase in the active region is favorable for interaction. (ii) Structural differences in small molecules: The enhancement in the surface area of Alisol G caused by the addition of Fe3+ is relatively more than by adding Cu2+, resulting in better spreading of the structure of Alisol G with Fe3+.
Moreover, the binding distance between Fe3+ and small molecules is shorter as compared with Cu2+, and the degree of polarization of Fe3+ is stronger than that of Cu2+, which results in a stronger deformation degree of DNA electron cloud in the active region, leading to the more probable overlap of the positive and negative electron clouds. The comparison of the binding positions of Fe3+, Cu2+, and Alisol G revealed that the higher valence metal ions were more susceptible to the action of the side chain hydroxyl groups of small molecules, whereas the lower valence metal ions were more susceptible to the action of the parent ring carbonyl group. Furthermore, the anticancer activity of Alisol G obtained after adding Fe3+ was better than upon Cu2+ addition, indicating that the side chain hydroxyl activity of Alisol G was superior to that of the parent ring carbonyl group activity. Thus, the side chain hydroxyl appeared as one of the key groups in the anticancer initiation of metal drugs.
The results of molecular dynamics simulation showed that the addition of metal ions did not affect the kinetic stability of the binding of Alisol G to c-myc DNA. The identified binding site of Cu2+ and Fe3+ in c-myc DNA was DG113. After the addition of metal ions, the binding domain of Alisol G in c-myc DNA changed, which promoted the intercalation combination of Alisol G and A-T base pair.
In this paper, due to the limitation of time, we mainly focused our efforts on studying the binding of Alisol G and c-myc DNA before and after the addition of metal ions. The mechanism and process of drug molecules interacting with other molecules in cells, including proteins, are very complex. According to the past investigations of our research group and the literature reviewed (Xu et al., 2020; Lu et al., 2021), Alisol G also binds to some important proteins in cancer cells and affects the cells. In-depth research in this direction is planned for our future work.
4 Conclusion
In pursuit of finding a potential anticancer drug derived from Chinese medicine, the effect of the addition of Cu2+ and Fe3+ on Alisol G, an active component of Alisma orientalis, was investigated in hepatoma cells. The results manifested that metal ions Cu2+ and Fe3+ can effectively inhibit the growth of liver cancer cells by promoting the binding between Alisol G and c-myc DNA. The binding ability between Alisol G and c-myc DNA was directly related to the type and position of its substituents under the action of metal ions, which indicated that its anticancer activity could be enhanced by improving its structure. The results provide ideas for exploring the molecular design and development of new metal-based traditional Chinese medicine anticancer drugs and showcase a promising treatment strategy for liver cancer.
Acknowledgements
This work was financially supported by the Jiangsu Provincial Bureau of Traditional Chinese Medicine Science and Technology Project (YB2017004), the Project of Industry-University- Research of Jiangsu Province (BY2018304), the National Nature Science Foundation of China (81303173, 81673534, 82073958), the Natural Science Foundation of Jiangsu Province (BK20161576), the Jiangsu Provincial Administration of Traditional Chinese Medicine Research Project (ZX2016D1).
References
Appendix A
Supplementary material
Supplementary data to this article can be found online at https://doi.org/10.1016/j.arabjc.2021.103425.
Appendix A
Supplementary material
The following are the Supplementary data to this article: