Evaluation of potential anti-metastatic and antioxidative abilities of natural peptides derived from Tecoma stans (L.) Juss. ex Kunth in A549 cells

Abstract

Background: Tecoma stans (L.) Juss. ex Kunth is a well-known medicinal plant found in tropical and subtropical regions. It contains a broad range of bioactive compounds that exhibit many biological effects, including antidiabetic, antibacterial, and antioxidative activities. However, the effect of natural peptides from T. stans against cancer progression and free radical production is unknown. This study aims to evaluate the cytotoxic, anti-metastatic, and antioxidative activities of natural peptides from T. stans on A549 cells.

Methods: The natural peptides were extracted from the flower of T. stans using the pressurized hot water extraction (PHWE) method, followed by size exclusion chromatography and solid-phase extraction-C18. The cytotoxic and anti-metastatic effects of natural peptides were evaluated using MTT and transwell chamber assays, respectively. The free radical scavenging activity of natural peptides was determined using ABTS, DPPH, and FRAP assays. The cells were pretreated with the IC₅₀ dosage of natural peptides and stimulated with LPS before analyzing intracellular reactive oxygen species (ROS) and proteomics.

Results: Natural peptides induced cell toxicity at a concentration of less than 1 ng/ml and markedly reduced cell motility of A549 cells. The cells had a migration rate of less than 10% and lost their invasion ability in the treatment condition. In addition, natural peptides showed free radical scavenging activity similar to standard antioxidants and significantly decreased intracellular ROS in the LPS-induced cells. Proteomic analysis revealed 1,604 differentially expressed proteins. The self-organizing tree algorithm (SOTA) clustered the protein abundances into eleven groups. The volcano plot revealed that the cancer-promoting proteins (NCBP2, AMD, MER34, ENC1, and COA4) were down-regulated, while the secretory glycoprotein (A1BG) and ROS-reducing protein (ASB6) were up-regulated in the treatment group.

Conclusion: The anti-proliferative and anti-metastatic activities of natural peptides may be attributed to the suppression of several cancer-promoting proteins. In contrast, their antioxidative activity may result from the up-regulation of ROS-reducing protein. This finding suggests that natural peptides from T. stans are viable for being the new potential anti-cancer and antioxidative agents.

Keywords: Anti-metastasis; Antioxidant; Medicinal plant; Natural peptide; Proteomics.

Figure 1. Stacked LC-MS/MS chromatograms of the natural peptides from T. stans.

(A) The flower of T. stans before extraction by PHWE method for obtaining natural peptides. (B) The chromatogram of natural peptides analyzed by LC-MS/MS including TIC (total ion count), MS1 spectrum, and MS2 spectrum. Each chromatogram has been shown in ions count vs retention time.

Figure 2. Natural peptides from T. stans showed selective cytotoxicity for human cancer cells.

The human cancer (A) A549, (B) HepG2, (C) HeLa, (D) SK-MEL-28, and (E) MCF-7 cells were treated with 0.0625, 0.125, 0.25, 0.5, 1.0 and 2.0 ng/ml of natural peptides, while the immortalized human keratinocyte (F) HaCat cells were treated with 0.391, 0.781, 1.563, 3.125, 6.25, 12.5 and 25.0 ng/ml of natural peptides for 24 h before monitoring cell viability using MTT assay. The results were obtained from three independent experiments, each in triplicate.

Figure 3. Natural peptides from T. stans markedly inhibited cell migration and invasion.

(A) Migration (left panels) and invasion (right panels) assays of A549 cells treated with or without 0.0625 ng/ml of natural peptides for 24 h. The migrated and invaded cells were stained with crystal violet. Scale bar, 100 µm. (B) The percentage of migration and invasion of A549 cells treated with natural peptides. The cells were counted from five random fields for each condition in three independent experiments. The percentage of migrated cells treated with natural peptides was relative to the untreated control. The percentage of invaded cells treated with or without natural peptides was relative to that of the migratory control cells that were allowed to migrate across the uncoated-Matrigel membrane. The statistical analysis was conducted using one-way ANOVA. Asterisks (***) indicate p-value of <0.001.

Figure 4. Natural peptides from T. stans exhibited antioxidative activity.

(A) DPPH, (B) ABTS and (C) FRAP assays were performed to evaluate the free radical scavenging capacity of natural peptides in comparison with ascorbic acid, gallic acid, and FeSO₄, respectively. (D) Intracellular ROS assay in the LPS-induced A549 cells. A549 cells were pretreated with or without 0.3321 ng/ml of natural peptides for 6 h, followed by 0.5 µg/ml LPS for 24 h before analyzing intracellular ROS. The data is represented as a mean ± SD from three independent experiments. The statistical analysis was conducted using paired Student’s t-test. Asterisks (***) indicate p-value <0.001).

Figure 5. Proteomic analysis of A549 cells treated with natural peptides from T. stans.

(A) SOTA dendrogram of protein abundance by sample. The heat-map shows the similarity of protein abundance in control and treatment groups. The differentially expressed proteins were grouped into 11 clusters with different protein expression profiles using centroid distance. (B) Volcano plot showing differential protein expressions. The points indicate different proteins that display both large magnitude fold changes (log₂ fold change, x-axis) and high statistical significance (-log10 of ANOVA, y-axis). The dashed horizontal line shows the cutoff values for ANOVA <0.001. The two vertical dashed lines indicate the fold change >2 in down-regulated (blue region) and up-regulated (pink region) proteins.