Polyphenolic Fraction Obtained From Thalassia testudinum Marine Plant and Thalassiolin B Exert Cytotoxic Effects in Colorectal Cancer Cells and Arrest Tumor Progression in a Xenograft Mouse Model

Abstract

Marine plants are important sources of pharmacologically active metabolites. The aim of the present work was to evaluate the cytotoxic and antitumor activity of a polyphenolic fraction obtained from Thalassia testudinum marine plant and thalassiolin B in human colorectal cancer cells. Human cancer cell lines, including HCT15, HCT116, SW260, and HT29 were treated with tested products for cytotoxicity evaluation by crystal violet assay. The potential proapoptotic effect of these natural products was assessed by flow cytometry in HCT15 cells at 48 h using Annexin V-FITC/propidium iodide. In addition, reactive oxygen species (ROS) generation was measured by fluorescence using DCFH-DA staining, and sulfhydryl concentration by spectrophotometry. The in vivo antitumor activity of the polyphenolic fraction (25 mg/kg) was evaluated in a xenograft model in nu/nu mice. In vivo proapoptotic effect was also evaluated by immunohistochemistry using anti-caspase 3 and anti-Bcl-2 antibodies. The results showed that tested products exert colorectal cancer cell cytotoxicity. Besides, the tested products induced a significant increase (p < 0.05) of intracellular ROS generation, and a depletion of sulfhydryl concentration in HCT15 cells. The polyphenolic fraction arrested tumor growth and induced apoptosis in the xenograft mice model. These results demonstrate the cytotoxic activity of T. testudinum metabolites associated, at least, with ROS overproduction and pro-apoptotic effects. Here we demonstrated for the first time the antitumor activity of a T. testudinum polar extract in a xenograft mice model. These results suggest the potential use of T. testudinum marine plant metabolites as adjuvant treatment in cancer therapy.

Keywords: T. testudinum; apoptosis; colorectal cancer; polyphenols; reactive oxygen species; thalassiolin B.

FIGURE 1

Chromatographic profile of thalassiolin B isolated from T. testudinum hydroethanolic extract. (A) Chemical structure of thalassiolin B (chrysoeriol 7-β-D-glucopyranosyl-2"-sulphate), the main component of T. testudinum extract. (B) HPLC of thalassiolin B standard. (C) HPLC profile of T. testudinum hydroethanolic extract. Adapted from: Garateix et al., 2011. The authors have the rights to share this figure.

FIGURE 2

Apoptosis of HCT15 cells after 48 h of exposure to the polyphenolic fraction and thalassiolin B obtained from T. testudinum extract (at IC₅₀ values). (A) Representative histogram of flow cytometry analysis by annexin V-FITC/propidium iodide staining. (B) Analysis of the percentage of apoptotic cells in each group. Cisplatin (10 µM) was used as positive control. PF, polyphenolic fraction; TB, thalassiolin (B). Bars represent the mean ± standard deviation of two independent experiments by triplicate. Different symbols represent statistical differences between groups (one-way ANOVA, followed by Tukey post-hoc test, p < 0.05).

FIGURE 3

Intracellular reactive oxygen species generation and sulfhydryl levels in HCT15 cells after 24 h of exposure to the polyphenolic fraction and thalassiolin B from T. testudinum extract (at IC₅₀ values). (A) control cells; (B): cisplatin (10 µM); (C): polyphenolic fraction; (D): thalassiolin B; (E): N-acetylcysteine + polyphenolic fraction; (F): N-acetylcysteine + thalassiolin (B). Bars represent mean ± standard deviation of fluorescence arbitrary units (G,H). Two independent experiments by triplicate were carried out. Different symbols on bars represent statistical differences (ANOVA, Tukey post-hoc, p < 0.05).

FIGURE 4

Toxicity assay of PF and TB in nu/nu female mice. (A): representative slides of H&E statining of different organs including liver, spleen and kidney. (B): time-course of body weight in different groups. Group 1: control (PBS, i.p.); group 2: cisplatin (2 mg/kg, i.p.); group 3: PF (25 mg/kg, i.p.); group 4: PF (250 mg/kg, i.p.); group 5: TB (25 mg/kg, i.p.); group 6: TB (250 mg/kg, i.p.). Bars: 50 µm, magnification ×20.

FIGURE 5

Antitumor effect of the polyphenolic fraction from T. testudinum extract in nu/nu mice. Animals (n = 5) were orally treated for 27 days with the polyphenolic fraction (25 mg/kg), or intraperitoneally with cisplatin (4 mg/kg) after xenograft model of HCT15 was established (A). Control group: animals treated with oral doses of PBS. Representative tumoral mass after mice’s euthanasia (B). Mean ± standard deviation of tumor volume and weight (E,F). Survival rate and body weight (G) behavior during the experiment (C,D).

FIGURE 6

Caspase 3 and Bcl-2 expression in tumoral tissue of nu/nu mice. Representative slides of cleaved caspase 3 expression and Bcl-2 in tumoral tissue determined by immunohistochemistry technique (five animals per group). Bar graphs represent the densitometry analysis of cleaved caspase 3 positive area/total area (%) and Bcl-2 positive area/total area (%). Bars: 50 µm, magnification ×20. Bars in the graphs represent the mean ± standard deviation of percentages. Asterisks represent statistical differences between groups (one-way ANOVA, followed by Tukey post-hoc test, p < 0.05).