Peptide of Trichinella spiralis Infective Larval Extract That Harnesses Growth of Human Hepatoma Cells

Abstract

Trichinella spiralis, a tissue-dwelling helminth, causes human trichinellosis through ingestion of undercooked meat containing the parasite's infective larvae. However, benefits from T. spiralis infection have been documented: reduction of allergic diseases, inhibition of collagen-induced arthritis, delay of type 1 diabetes progression, and suppression of cancer cell proliferation. Since conventional cancer treatments have limited and unreliable efficacies with adverse side effects, novel adjunctive therapeutic agents and strategies are needed to enhance the overall treatment outcomes. This study aimed to validate the antitumor activity of T. spiralis infective larval extract (LE) and extricate the parasite-derived antitumor peptide. Extracts of T. spiralis infective larvae harvested from striated muscles of infected mice were prepared and tested for antitumor activity against three types of carcinoma cells: hepatocellular carcinoma HepG2, ovarian cancer SK-OV-3, and lung adenocarcinoma A549. The results showed that LE exerted the greatest antitumor effect on HepG2 cells. Proteomic analysis of the LE revealed 270 proteins. They were classified as cellular components, proteins involved in metabolic processes, and proteins with diverse biological functions. STRING analysis showed that most LE proteins were interconnected and played pivotal roles in various metabolic processes. In silico analysis of anticancer peptides identified three candidates. Antitumor peptide 2 matched the hypothetical protein T01_4238 of T. spiralis and showed a dose-dependent anti-HepG2 effect, not by causing apoptosis or necrosis but by inducing ROS accumulation, leading to inhibition of cell proliferation. The data indicate the potential application of LE-derived antitumor peptide as a complementary agent for human hepatoma treatment.

Keywords: Trichinella spiralis; antitumor peptide; drug discovery; human hepatocellular carcinoma HepG2 cell; infective larva; proteomics.

Figure 1

Antitumor activity of the T. spiralis infective larval extracts (LE). HepG2, SK-OV-3, and A549 cells (5 × 10³ cells) in individual wells of a 96-well culture plate were treated with 70 µgmL^-1 of T. spiralis LE for 24 h. (A) Percentage cell survival of HepG2 cells at 24 h posttreatment with different concentrations of PIC. (B) Morphological alteration and proliferation of HepG2 cells at 24 h after treatment with different concentrations of PIC visualized at 100× microscopic magnification. (C) Percentage cell survivals of HepG2, SK-OV-3, and A549 cells at 24 h posttreatment with 70 µgmL^-1 of LE compared with the control (cells treated with medium supplemented with 0.05% PIC). The results are shown as the mean ± standard deviation (SD) of three independent experiments. ^{∗ ∗} P < 0.01; ^{∗ ∗ ∗} P < 0.001.

Figure 2

Cytotoxic effects of the infective larval extract (LE) of T. spiralis on human hepatocellular carcinoma HepG2 cells. (A) Preparation of the LE from T. spiralis infective larvae collected from infected mice. (B) Percentage cell survivals of HepG2 cells at 24 h posttreatment with different concentrations (8.75, 17.5, 35, and 70 µgmL^-1) of LE. The results are shown as the mean ± standard deviation (SD) of three independent experiments. ^∗ P < 0.05; ^{∗ ∗} P < 0.01; ^{∗ ∗ ∗} P < 0.001. (C) Proliferations and morphological changes in HepG2 cells treated with LE (70 µgmL^-1) compared with untreated cells at 24 h. The cells were visualized microscopically at 200× magnification.

Figure 3

Protein content of the T. spiralis infective larval extract (LE). (A) Flow diagram of proteomic analysis of LE using electrospray ionization quadrupole ion mobility time-of-flight mass spectrometry (ESI-QUAD-TOF) and OmicsBox software. (B) Protein patterns of the LEs on a 12% gel after SDS-PAGE and CBB staining. M, protein marker; numbers on the left are protein molecular masses in kDa. (C) Two-dimensional protein relative molecular masses (Mr) and the pH value at which the total charge on each protein was zero (pI).

Figure 4

Protein-protein interaction network of identified proteins of the muscle larval extract (LE) of Trichinella spiralis using the STRING database. The types of interactions are represented by different colored lines (lower left panel). The functions of each protein-encoding gene are represented by different colored circle lines (lower right panel).

Figure 5

Ribbon diagram of the selected antitumor peptide candidates. Amino acid sequences of the three peptide candidates were predicted for their peptide structures using PEP-FOLD3 (Lamiable et al., 2016). (A) Predicted peptide structure (upper panel) with the local structure prediction profile (lower panel) of antitumor peptide 1. (B) Predicted peptide structure with the local structure prediction profile of antitumor peptide 2. (C) Predicted peptide structure with the local structure prediction profile of antitumor peptide 3. The profile is presented using the following color codes: red, helical; green, extended; and blue, coil.

Figure 6

Antihepatocellular carcinoma activity of the three peptide candidates. HepG2 cell lines (5 × 10³ cells) in individual wells of a 96-well culture plate were treated with different concentrations (5.5, 11, 22, 44, 88, 176, and 352 µgmL^-1) of individual antitumor peptides for 24 h. (A) Percentage cell survivals of HepG2 cells at 24 h posttreatment with different concentrations of antitumor peptide 1. (B) Percentage cell survivals of HepG2 cells at 24 h posttreatment with different concentrations of antitumor peptide 2. (C) Percentage cell survivals of HepG2 cells at 24 h posttreatment with different concentrations of antitumor peptide 3. Medium alone and medium supplemented with 1% DMSO were used as negative and diluent controls, respectively. The results are shown as the mean ± standard deviation (SD) of three independent experiments. ^∗ P < 0.05; ^{∗ ∗} P < 0.01; ^{∗ ∗ ∗} P < 0.001.

Figure 7

Analysis of cancer cell apoptosis and necrosis mediated by antitumor peptide 2. HepG2 cells (5 × 10⁵ cells) were treated with 337 µgmL^-1 of antitumor peptide 2, 1% DMSO (diluent control), and medium alone (negative control) for 24 h. Cells of all treatments were stained with fluorochrome-conjugated Annexin V and propidium iodide (PI) and analyzed by flow cytometry. (A) FACS analyses of Annexin V- and PI-stained HepG2 cells treated with antitumor peptide 2. (B) FACS analyses of Annexin V- and PI-stained HepG2 cells treated with 1% DMSO. (C) FACS analyses of Annexin V- and PI-stained HepG2 cells treated with medium alone. Lower right quadrant (Q3) Annexin V-positive/PI-negative cells denote early apoptotic cells, and upper right quadrant (Q2) Annexin V-positive/PI-positive cells denote necrotic or late apoptotic cells.

Figure 8

Production of reactive oxygen species (ROS) within HepG2 cells after antitumor peptide 2 treatment. HepG2 cells (1 × 10⁵ cells) were treated with 337 µgmL^-1 of antitumor peptide 2, 50 µM of tert-butyl hydrogen peroxide (tbHP; positive control), medium alone (negative control), and 1% DMSO (diluent control) for 24 h. All cells were stained with 2’, 7’-dichlorofluorescin diacetate (DCFDA) solution (DCFDA/H2DCFDA-Cellular ROS Assay Kit) and visualized by confocal microscopy at 400× magnification. Images were recorded under FITC, TD, and all channels. Scale bar = 50 μm.