In Vitro and In Vivo Effects of Ulvan Polysaccharides from Ulva rigida

Abstract

One of the main bioactive compounds of interest from the Ulva species is the sulfated polysaccharide ulvan, which has recently attracted attention for its anticancer properties. This study investigated the cytotoxic activity of ulvan polysaccharides obtained from Ulva rigida in the following scenarios: (i) in vitro against healthy and carcinogenic cell lines (1064sk (human fibroblasts), HACAT (immortalized human keratinocytes), U-937 (a human leukemia cell line), G-361 (a human malignant melanoma), and HCT-116 (a colon cancer cell line)) and (ii) in vivo against zebrafish embryos. Ulvan exhibited cytotoxic effects on the three human cancer cell lines tested. However, only HCT-116 demonstrated sufficient sensitivity to this ulvan to make it relevant as a potential anticancer treatment, presenting an LC₅₀ of 0.1 mg mL^-1. The in vivo assay on the zebrafish embryos showed a linear relationship between the polysaccharide concentration and growth retardation at 7.8 hpf mL mg^-1, with an LC₅₀ of about 5.2 mg mL^-1 at 48 hpf. At concentrations near the LC₅₀, toxic effects, such as pericardial edema or chorion lysis, could be found in the experimental larvae. Our in vitro study supports the potential use of polysaccharides extracted from U. rigida as candidates for treating human colon cancer. However, the in vivo assay on zebrafish indicated that the potential use of ulvan as a promising, safe compound should be limited to specific concentrations below 0.001 mg mL^-1 since it revealed side effects on the embryonic growth rate and osmolar balance.

Keywords: Ulva rigida; cytotoxic activity; human cancer cell lines; polysaccharides; ulvan; zebrafish embryo toxicity test.

Figure 1

FTIR spectroscopy of ulvans from U. rigida.

Figure 2

Gas chromatography–mass spectrometry (GC–MS) of ulvans from U. rigida.

Figure 3

Cytotoxic activity of ulvan polysaccharides, expressed as survival (%) of each cell line depending on the ulvan polysaccharide concentration (mg mL⁻¹). Each figure represents a cell line: (a) immortalized human keratinocytes (HACAT), (b) human fibroblasts (1064SK), (c) human colorectal carcinoma cell line (HCT-116), (d) human myeloid leukemia (U-937), and (e) human malignant melanoma (G-361).

Figure 4

Standard length and head–trunk angle of zebrafish embryos. (A) Control untreated zebrafish embryos. The double discontinuous arrow shows the standard length (SL). (B) Polysaccharide-treated 72 hpf embryo. α indicates the head–trunk angle as suggested by Kimmel et al. [60]. Bars represent 500 µm.

Figure 5

LC₅₀ estimation for ulvan polysaccharide treatment of zebrafish 48 hpf embryos. Circles and vertical bars are the means and standard deviations of data from three replicates. Gray, transparent circles are data used to estimate the LC₅₀. The empty circle intersects the regression line and 50 % viability. The white circle is the log (LC₅₀) estimation. Linear adjustment is y = −7.2237x + 5.6279 (R² = 0.8357).

Figure 6

Linear functions used to transform standard lengths and head–trunk angles into hours of post-fertilization development. Graphs show the linear adjustments recovered from approximations to standard length (A) and head–trunk angle (B) data in Kimmel et al. [60] (Figures 16 and 33, respectively, of Kimmel et al. [60]). Data were obtained using ImageJ 1.50i (nih.gov, accessed on 13 January 2023). The variable transformation functions are (A) y = 0.0207x + 2.0153 (R² = 0.8716; p ≈ 0.0000) (greater sizes); y = 0.1124x − 0.9848 (R² = 0.931; p ≈ 0.0000) (lesser sizes); and (B) y = 0.8216x + 97.927 (R² = 0.6126; p < 0.00059) (greater angles); y = 3.2744x − 12.452 (R² = 0.9081; p < 0.000835) (lesser angles). Discontinuous lines represent the variable transformation limits.

Figure 7

Embryo stage estimation after anatomical variable transformation using data from Kimmel et al. [60] (see Figure 4). (A–C) Linear regressions of standard length (A), head–trunk α angle (B), and anatomical-based estimations (C) with respect to the ulvan concentration. (D) Linear reduction in the compound stage estimation (A + B + C) with respect to the ulvan concentration. The linear adjustments are (A) y = −7.2x + 66.234 (R² = 0.7232; p ≈ 0.0000), (B) y = −7.3294x + 76.026 (R² = 0.8003; p < 0.00015), (C) y = −8.8x + 70.857 (R² = 0.8738; p ≈ 0.0000), and (D) y = −7.88x + 71.036 (R² = 0.7479; p < 0.000085).

Figure 8

Pericardial edema and chorion lysis increase with the ulvan polysaccharide concentration. Slight (A) and significant (B) pericardial edemas are seen in zebrafish embryos treated with a 0.25 mg mL⁻¹ ulvan concentration. (C) Exponential regression of edema frequency versus ulvan polysaccharide concentrations (y = 0.0386x^0.2325, R² = 0.9351). (D) Chorion lysis observed at 2.5 mg mL⁻¹ ulvan. Bars represent 500 µm.