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

Abstract

The carnivorous pitcher plants of the genus Nepenthes have long been known for their ethnobotanical applications. In this study, we prepared various extracts from the pitcher, stem, and leaf of Nepenthes miranda using 100% ethanol and assessed their inhibitory effects on key enzymes related to skin aging, including elastase, tyrosinase, and hyaluronidase. The cytotoxicity of the stem extract of N. miranda on H838 human lung carcinoma cells were also characterized by effects on cell survival, migration, proliferation, apoptosis induction, and DNA damage. The cytotoxic efficacy of the extract was enhanced when combined with the chemotherapeutic agent 5-fluorouracil (5-FU), indicating a synergistic effect. Flow cytometry analysis suggested that the stem extract might suppress H838 cell proliferation by inducing G2 cell cycle arrest, thereby inhibiting carcinoma cell proliferation. Gas chromatography-mass spectrometry (GC-MS) enabled the tentative identification of the 15 most abundant compounds in the stem extract of N. miranda. Notably, the extract showed a potent inhibition of the human RPA32 protein (huRPA32), critical for DNA replication, suggesting a novel mechanism for its anticancer action. Molecular docking studies further substantiated the interaction between the extract and huRPA32, highlighting bioactive compounds, especially the two most abundant constituents, stigmast-5-en-3-ol and plumbagin, as potential inhibitors of huRPA32's DNA-binding activity, offering promising avenues for cancer therapy. Overall, our findings position the stem extract of N. miranda as a promising source of natural compounds for anticancer therapeutics and anti-skin-aging treatments, warranting further investigation into its molecular mechanisms and potential clinical applications.

Keywords: AntoDock; GC–MS analysis; H838 lung carcinoma; Nepenthes; RPA; anti-skin aging; anticancer; elastase; plumbagin; stigmast-5-en-3-ol.

Figure 1

Inhibition of elastase activity by N. miranda extract. The inhibitory effects were demonstrated on elastase activity by (A) EGCG and N. miranda extracts from the (B) pitcher, (C) leaf, and (D) 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 N. miranda extract). Levels of statistical significance are denoted by * p < 0.05, ** p < 0.01, and *** p < 0.001 when compared to the control group.

Figure 2

Inhibition of tyrosinase activity by N. miranda extract. The inhibitory effects were investigated on tyrosinase activity by (A) KA, (B) Que, and N. miranda extracts from the (C) pitcher, (D) leaf, and (E) 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 N. miranda extract). Levels of statistical significance are indicated by * p < 0.05, ** p < 0.01, and *** p < 0.001 in comparison to the control group.

Figure 3

Inhibition of hyaluronidase activity by N. miranda extract. The inhibitory effects were investigated on hyaluronidase activity by (A) Myr, and N. miranda extracts from the (B) pitcher, (C) leaf, and (D) 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 N. miranda extract). Levels of statistical significance are indicated by ** p < 0.01 and *** p < 0.001 in comparison to the control group.

Figure 4

Anticancer potential of N. miranda stem extract on H838 cells. (A) The effect of N. miranda stem extract on H838 cell survival, migration, proliferation, and nuclear condensation. (B) Trypan blue exclusion assay showing H838 cell viability following treatment with various concentrations of N. miranda extract. (C) Wound healing assay depicting the migration of H838 cells treated with different concentrations of N. miranda extract. Images were captured immediately and 24 h post-treatment. (D) Clonogenic assay assessing the proliferative and colony-forming potential of H838 cells pre-treated with varying concentrations of N. miranda extract. (E) Hoechst staining illustrating apoptosis and DNA fragmentation in H838 cells at varying concentrations of N. miranda extract. Statistical significance is denoted by ** p < 0.01 and *** p < 0.001 when compared to the control group.

Figure 5

DNA damage induced in H838 cells by N. miranda extract. (A) Comet assay results display a significant escalation in DNA damage as the concentration of N. miranda extract increases. (B) A marked increase in comet tail density and (C) an extension in comet tail length were observed, reflective of a concentration-dependent rise in DNA damage. Levels of statistical significance are indicated by ** p < 0.01 and *** p < 0.001 in comparison to the control group. ns—non-significant.

Figure 6

(A) Alteration of cell cycle progression by N. miranda stem extract in H838 cells. H838 cells underwent treatment with a control solution (0.1% DMSO) or with N. miranda 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) The cell cycle distribution.

Figure 7

Synergistic anticancer effects of N. miranda stem extract and 5-FU on H838 Cells. The combined impact of N. miranda 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 N. miranda stem extract, may enhance therapeutic efficacy against cancer.

Figure 8

Inhibition of the ssDNA-binding activity of huRPA32 by different extracts of N. miranda. (A) 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]₅₀) 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 N. miranda extracts (0, 0, 7.6, 15.1, 31.3, 62.5, 125, 250, 500, 1000 μg/mL) from the (B) pitcher, (C) leaf, and (D) stem. An amount of 1% DMSO was used as a negative control (representing 0 μg/mL of N. miranda extract). The “w/o” denotes the absence of huRPA32 and the extract during the incubation with the DNA dT25.

Figure 9

Molecular docking analysis of huRPA32. (A) 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) 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. (C) 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. (D) 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. (E) The binding mode of plumbagin, which formed a hydrogen bond with His131 and engaged in π-stacking with Phe64. (F) 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.