Cytotoxicity and Multi-Enzyme Inhibition of Nepenthes miranda Stem Extract on H838 Human Non-Small Cell Lung Cancer Cells and RPA32, Elastase, Tyrosinase, and Hyaluronidase Proteins
<p>Inhibition of elastase activity by <span class="html-italic">N. miranda</span> extract. The inhibitory effects were demonstrated on elastase activity by (<b>A</b>) EGCG and <span class="html-italic">N. miranda</span> extracts from the (<b>B</b>) pitcher, (<b>C</b>) leaf, and (<b>D</b>) stem. AAAPVN was utilized as the substrate. EGCG was employed as a positive control, while 10% DMSO was used as a negative control (indicating 0 μg/mL of <span class="html-italic">N. miranda</span> extract). Levels of statistical significance are denoted by * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01, and *** <span class="html-italic">p</span> < 0.001 when compared to the control group.</p> "> Figure 2
<p>Inhibition of tyrosinase activity by <span class="html-italic">N. miranda</span> extract. The inhibitory effects were investigated on tyrosinase activity by (<b>A</b>) KA, (<b>B</b>) Que, and <span class="html-italic">N. miranda</span> extracts from the (<b>C</b>) pitcher, (<b>D</b>) leaf, and (<b>E</b>) stem. L-DOPA was employed as the substrate. KA and Que were utilized as positive controls, while 10% DMSO was used as a negative control (representing 0 μg/mL of <span class="html-italic">N. miranda</span> extract). Levels of statistical significance are indicated by * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01, and *** <span class="html-italic">p</span> < 0.001 in comparison to the control group.</p> "> Figure 3
<p>Inhibition of hyaluronidase activity by <span class="html-italic">N. miranda</span> extract. The inhibitory effects were investigated on hyaluronidase activity by (<b>A</b>) Myr, and <span class="html-italic">N. miranda</span> extracts from the (<b>B</b>) pitcher, (<b>C</b>) leaf, and (<b>D</b>) stem. Hyaluronic acid was employed as the substrate. Myr was utilized as a positive control, while 10% DMSO was used as a negative control (representing 0 μg/mL of <span class="html-italic">N. miranda</span> extract). Levels of statistical significance are indicated by ** <span class="html-italic">p</span> < 0.01 and *** <span class="html-italic">p</span> < 0.001 in comparison to the control group.</p> "> Figure 4
<p>Anticancer potential of <span class="html-italic">N. miranda</span> stem extract on H838 cells. (<b>A</b>) The effect of <span class="html-italic">N. miranda</span> stem extract on H838 cell survival, migration, proliferation, and nuclear condensation. (<b>B</b>) Trypan blue exclusion assay showing H838 cell viability following treatment with various concentrations of <span class="html-italic">N. miranda</span> extract. (<b>C</b>) Wound healing assay depicting the migration of H838 cells treated with different concentrations of <span class="html-italic">N. miranda</span> extract. Images were captured immediately and 24 h post-treatment. (<b>D</b>) Clonogenic assay assessing the proliferative and colony-forming potential of H838 cells pre-treated with varying concentrations of <span class="html-italic">N. miranda</span> extract. (<b>E</b>) Hoechst staining illustrating apoptosis and DNA fragmentation in H838 cells at varying concentrations of <span class="html-italic">N. miranda</span> extract. Statistical significance is denoted by ** <span class="html-italic">p</span> < 0.01 and *** <span class="html-italic">p</span> < 0.001 when compared to the control group.</p> "> Figure 5
<p>DNA damage induced in H838 cells by <span class="html-italic">N. miranda</span> extract. (<b>A</b>) Comet assay results display a significant escalation in DNA damage as the concentration of <span class="html-italic">N. miranda</span> extract increases. (<b>B</b>) A marked increase in comet tail density and (<b>C</b>) an extension in comet tail length were observed, reflective of a concentration-dependent rise in DNA damage. Levels of statistical significance are indicated by ** <span class="html-italic">p</span> < 0.01 and *** <span class="html-italic">p</span> < 0.001 in comparison to the control group. ns—non-significant.</p> "> Figure 6
<p>(<b>A</b>) Alteration of cell cycle progression by <span class="html-italic">N. miranda</span> stem extract in H838 cells. H838 cells underwent treatment with a control solution (0.1% DMSO) or with <span class="html-italic">N. miranda</span> stem extract at specified concentrations for 24 h and were subsequently fixed in 70% ethanol overnight. The cells were then stained with propidium iodide (PI) for 30 min before analysis via flow cytometry. (<b>B</b>) The cell cycle distribution.</p> "> Figure 7
<p>Synergistic anticancer effects of <span class="html-italic">N. miranda</span> stem extract and 5-FU on H838 Cells. The combined impact of <span class="html-italic">N. miranda</span> stem extract (20 μg/mL) and 5-FU (5 μM) on the survival, migration, proliferation, and apoptosis of H838 cells was assessed. Evaluations were conducted using trypan blue dye exclusion staining for cell viability, Hoechst staining for apoptosis detection, a wound-healing assay for migration analysis, and a clonogenic assay for proliferative capacity. The outcomes of the combination treatment suggest that 5-FU, when used alongside <span class="html-italic">N. miranda</span> stem extract, may enhance therapeutic efficacy against cancer.</p> "> Figure 8
<p>Inhibition of the ssDNA-binding activity of huRPA32 by different extracts of <span class="html-italic">N. miranda</span>. (<b>A</b>) Binding of huRPA32 to ssDNA dT25. Purified huRPA32 (0, 0.32, 0.63, 1.25, 2.5, 5, 7.5, 10, 20, and 40 μM) was incubated with biotin-labeled ssDNA dT25 and the binding was analyzed with EMSA. The binding constant ([Protein]<sub>50</sub>) of huRPA32 was quantified through linear interpolation based on the protein concentrations. The inhibitory effects were investigated on the DNA-binding activity of huRPA32 by <span class="html-italic">N. miranda</span> extracts (0, 0, 7.6, 15.1, 31.3, 62.5, 125, 250, 500, 1000 μg/mL) from the (<b>B</b>) pitcher, (<b>C</b>) leaf, and (<b>D</b>) stem. An amount of 1% DMSO was used as a negative control (representing 0 μg/mL of <span class="html-italic">N. miranda</span> extract). The “w/o” denotes the absence of huRPA32 and the extract during the incubation with the DNA dT25.</p> "> Figure 9
<p>Molecular docking analysis of huRPA32. (<b>A</b>) The crystal structure of huRPA32 in complex with huRPA70 and huRPA14 (PDB ID 1L1O), with huRPA70 colored blue, huRPA32 green, and huRPA14 light magenta. (<b>B</b>) The modeled huRPA–ssDNA complex, constructed by manually superimposing the apo-huRPA structure with the PaRPA complex (PDB ID 8AAS), assuming similar ssDNA binding mechanisms across species. The ssDNA from the PaRPA complex is colored yellow. (<b>C</b>) Docking analysis showing the seven most abundant compounds from the stem extract individually docked into huRPA32: stigmast-5-en-3-ol in hot pink, plumbagin in orange, hexadecanoic acid in purple-blue, hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl)ethyl ester in dark salmon, catechol in cyan, oleic acid in wheat, and pyrogallol in chocolate. Five of these seven compounds targeted various ssDNA binding sites in huRPA32, potentially collectively hindering the binding of ssDNA to huRPA32. The charge distribution pattern is shown to indicate ssDNA binding sites for clarity. (<b>D</b>) The binding mode of stigmast-5-en-3-ol, situated within the cavity of the ssDNA-binding surface, engaging in extensive hydrophobic interactions with Leu59, Glu62, Val63, Phe64, and Gln73 of huRPA32. (<b>E</b>) The binding mode of plumbagin, which formed a hydrogen bond with His131 and engaged in π-stacking with Phe64. (<b>F</b>) The binding mode of hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl)ethyl ester, anchored within the groove for ssDNA binding of huRPA32, formed hydrogen bonds with Gln106 and Trp107, and also interacted hydrophobically with Arg105, Val142, and Phe144.</p> ">
Abstract
:1. Introduction
2. Results
2.1. Anti-Elastase Activity of N. miranda Extract
2.2. Anti-Tyrosinase Activity of N. miranda Extract
2.3. Anti-Hyaluronidase Activity of N. miranda Extract
2.4. Cytotoxic Activity of the Ethanol Extract of Nepenthes miranda on H838 Lung Adenocarcinoma Cells
2.5. The Extract of N. miranda Inhibited the Migration of H838 Cells
2.6. The Extract of N. miranda Inhibited the Proliferation of H838 Cells
2.7. The Extract of N. miranda Induced Apoptosis of H838 Cells
2.8. The Extract of N. miranda Caused DNA Damage in H838 Cells
2.9. Gas Chromatography–Mass Spectrometry (GC–MS) Analysis
2.10. The Stem Extract of N. miranda Suppressed Carcinoma Cell Proliferation by Inducing G2 Cell-Cycle Arrest
2.11. Co-Treatment of N. miranda Stem Extract with the Anticancer Drug 5-Fluorouracil (5-FU) against H838 Cells
2.12. Inhibition of Human RPA32 by N. miranda Extracts
2.13. Molecular Docking of huRPA32
3. Discussion
4. Materials and Methods
4.1. Materials
4.2. Expression and Purification of the Recombinant Protein
4.3. Electrophoretic Mobility Shift Analysis (EMSA)
4.4. huRPA32 Inhibition
4.5. Plant Materials and Extract Preparations
4.6. GC-MS Analysis
4.7. Elastase Inhibition
4.8. Tyrosinase Inhibition
4.9. Hyaluronidase Inhibition
4.10. Trypan Blue Cytotoxicity Assay
4.11. Chromatin Condensation Assay
4.12. Clonogenic Formation Assay
4.13. Wound-Healing Assay
4.14. Flow Analysis
4.15. Comet Assay
4.16. Binding Analysis Using AutoDock Vina
4.17. Statistical Analysis
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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IC50 Value (μg/mL) | ||||
---|---|---|---|---|
Inhibitor | Elastase | Tyrosinase | Hyaluronidase | RPA32 |
Stem extract | 2.29 ± 0.68 | 48.33 ± 2.92 | 7.89 ± 0.64 | 101.6 ± 6.3 |
Leaf extract | 5.31 ± 0.31 | 180.57 ± 0.75 | 16.18 ± 1.03 | 231.4 ± 12.5 |
Pitcher extract | 8.66 ± 1.15 | ND | 31.67 ± 2.96 | 706.6 ± 32.6 |
KA | – | 3.94 ± 0.32 | – | – |
Que | – | 33.64 ± 2.00 | – | – |
EGCG | 4.41 ± 0.34 | – | – | – |
Myr | – | – | 9.52 ± 0.27 | – |
Affinity (kcal/mol) | Interaction | Residue (Distance, Å) | |
---|---|---|---|
Stigmast-5-en-3-ol | −7.2 | Hydrophobic | L59 (3.59), E62 (3.98), V63 (3.80), F64 (3.59, 3.83, 3.85), Q73 (3.54) |
Plumbagin | −6.2 | Hydrogen bond | H131 (3.75) |
π-Stacking | F64 (4.40) | ||
Hexadecanoic acid | −5.1 | Hydrophobic | T50 (3.57, 3.72, 3.92), V77 (4.00), D96 (3.66), T98 (3.42), F155 (3.50), H158 (3.65), V162 (3.62), I163 (3.77) |
Hexadecanoic acid, 2-hydroxy- | −5.3 | Hydrogen bond | Q106 (3.10, 3.86), W107 (3.00) |
1-(hydroxymethyl)ethyl ester | Hydrophobic | R105 (3.77), V142 (3.69, 3.73), F144 (3.62) | |
Catechol | −4.7 | Hydrogen bond | E62 (3.74), R133 (2.80), S134 (2.81) |
Hydrophobic | L59 (3.62, 3.74), F64 (3.60, 3.73) | ||
Oleic acid | −5.3 | Hydrogen bond | V77 (2.87), I159 (3.77) |
Hydrophobic | T50 (3.61), D96 (3.76), T98 (3.49), I159 (3.55, 3.64), I163 (3.61, 3.71), H166 (3.62) | ||
Pyrogallol | −4.8 | Hydrogen bond | V77 (2.89), D96 (2.94) |
Hydrophobic | T50 (3.73), I159 (3.53) |
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Lee, C.-Y.; Chen, Y.-C.; Huang, Y.-H.; Lien, Y.; Huang, C.-Y. Cytotoxicity and Multi-Enzyme Inhibition of Nepenthes miranda Stem Extract on H838 Human Non-Small Cell Lung Cancer Cells and RPA32, Elastase, Tyrosinase, and Hyaluronidase Proteins. Plants 2024, 13, 797. https://doi.org/10.3390/plants13060797
Lee C-Y, Chen Y-C, Huang Y-H, Lien Y, Huang C-Y. Cytotoxicity and Multi-Enzyme Inhibition of Nepenthes miranda Stem Extract on H838 Human Non-Small Cell Lung Cancer Cells and RPA32, Elastase, Tyrosinase, and Hyaluronidase Proteins. Plants. 2024; 13(6):797. https://doi.org/10.3390/plants13060797
Chicago/Turabian StyleLee, Ching-Yi, Yu-Cheng Chen, Yen-Hua Huang, Yi Lien, and Cheng-Yang Huang. 2024. "Cytotoxicity and Multi-Enzyme Inhibition of Nepenthes miranda Stem Extract on H838 Human Non-Small Cell Lung Cancer Cells and RPA32, Elastase, Tyrosinase, and Hyaluronidase Proteins" Plants 13, no. 6: 797. https://doi.org/10.3390/plants13060797