Antiproliferative, Antioxidant, Chemopreventive and Antiangiogenic Potential of Chromatographic Fractions from Anemonia sulcata with and without Its Symbiont Symbiodinium in Colorectal Cancer Therapy

Abstract

Anemonia sulcata may be a source of marine natural products (MNPs) due to the antioxidant and antitumor activity of its crude homogenates shown in vitro in colon cancer cells. A bioguided chromatographic fractionation assay of crude Anemonia sulcata homogenates with and without its symbiont Symbiodinium was performed to characterize their bioactive composition and further determine their biological potential for the management of colorectal cancer (CRC). The 20% fractions retained the in vitro antioxidant activity previously reported for homogenates. As such, activation of antioxidant and detoxifying enzymes was also evaluated. The 40% fractions showed the greatest antiproliferative activity in T84 cells, synergistic effects with 5-fluoruracil and oxaliplatin, overexpression of apoptosis-related proteins, cytotoxicity on tumorspheres, and antiangiogenic activity. The predominantly polar lipids and toxins tentatively identified in the 20% and 40% fractions could be related to their biological activity in colon cancer cells although further characterizations of the active fractions are necessary to isolate and purify the bioactive compounds.

Keywords: Anemonia sulcata; Symbiodinium; antiangiogenic activity; antioxidant activity; antitumor activity; chemopreventive activity; colorectal cancer; fractionation.

Figure 1

Antiproliferative activity of chromatographic fractions of crude homogenate from A. sulcata. T84 colorectal cancer cells were treated with three acetonitrile-eluted chromatographic fractions (20, 40, 60%) from (A) HOMG W and (B) W/O at a concentration range of 0.05 to 75 µg/mL for 72 h. Relative proliferation (%RP) was calculated as the mean ± standard deviation (SD) of 3 replicates from two different experiments measured by MTT colorimetric assay. Statistically significant differences compared to untreated cells were calculated: p value < 0.05 (*); <0.01 (**); <0.001 (***).

Figure 2

Heatmaps of T84 cells treated with crude homogenates or 40% fractions from A. sulcata in combination with (A) 5FU or (B) OXA. Different concentrations of HOMG W, W/O, or 40% W and 40% W/O were co-incubated with 5FU (0.08, 0.2 and 0.39 μg/mL, respectively) or OXA (0.3, 0.64, and 1.19 μg/mL, respectively) for 72 h. Synergy scores (HLA model) were represented in Heatmaps with SynergyFinder Plus, where areas of synergy (values >10 in red color), additive effect (from −10 to 10 in white color), and antagonism (<−10 in green color) were distinguished.

Figure 3

HCT116 tumorspheres formation, characterization, and treatment with 40% chromatographic fractions from A. sulcata. (A) Light microscopy images of tumorspheres after 10 days of induction. (B) Relative expression of CSC-related markers in HCT116 tumorspheres by RT-qPCR. (C) Viability assay of HCT116 tumorspheres treated with 40% W and W/O (400 μg/mL) using CCK8 assay. Results are expressed as % cell viability with respect to the total population of control cells. Statistically significant differences compared to untreated cells were calculated: p value < 0.001 (***).

Figure 4

Western blot analysis of (A) caspase 8, (B) caspase 9, (C) PARP1 protein expression from cells treated with HOMG W and W/O, and 40% W and W/O. The bands obtained by chemiluminescence were analyzed using Quantity One 4.6.8 analytical software, and relative expression (Protein/β-actin) was calculated as the mean ± SD of three measurements from three Western Blot replicates. Statistically significant differences compared to untreated cells were calculated: p value < 0.05 (*); <0.01 (**); <0.001 (***).

Figure 5

Study of the antiangiogenic potential of crude homogenates and 40% chromatographic fraction of A. sulcata. (A) Images of both treatment and external areas of CAM subjected to different conditions (15 μL/egg): negative control (saline solution), positive control (Aflibercept [Aflib], 10 mg/mL), HOMG W and W/O (0.5 mg/mL), and 40% W and W/O (0.5 mg/mL). Images were analyzed with the plugin “Vessel Analysis” from FIJI and the % area of (B) vascular density and (C) vascular length density were obtained for each condition. (D) Protein VEGFA expression from each condition was studied by Western Blot analysis. Bands obtained by chemiluminescence were analyzed using Quantity One analytical software, and relative expression (VEGFA/β-actin) was calculated as the mean ± SD of three measurements from three Western Blot replicates. Statistically significant differences compared to the negative control were calculated: p value < 0.05 (*); <0.01 (**); <0.001 (***).

Figure 6

Antioxidant activity of chromatographic fractions of A. sulcata in vitro. HT29 colorectal cancer cells were subjected to a 24 h pretreatment with two different non-cytotoxic concentrations of each of the fractions from HOMG W and W/O: (A) 20% W; (B) 20% W/O; (C) 40% W; (D) 40% W/O; (E) 60% W; and (F) 60% W/O. After this, the medium was changed, and cells were incubated for 6 h with two doses of H₂O₂ (1.68 and 1.85 mM). Relative proliferation (%RP) was obtained by MTT assay. Data are represented as the mean ± SD of 8 replicates. Statistically significant differences compared to cells treated with H₂O₂ only were calculated: p value < 0.001 (***).

Figure 7

(A) Catalase (CAT), (B) Superoxide dismutase (SOD), (C) Glutathione peroxidase (GPX), (D) Glutathione S-Transferase (GST), and (E) NAD(P)H: quinone oxidoreductase (QR) activity induced by cytosolic fractions from A. sulcata with and without symbiont homogenates and their respective 20% ACN fraction. Cytosolic fractions were obtained from cell cultures treated with HOMG W and W/O (0.5 µg/mL) and 20% W and W/O fractions (5 µg/mL) for 48 h. GST and QR activity was measured at 340 and 600 nm, respectively, and UA/mg protein and induction rate (treated/control) were calculated. Cells treated with sulforaphane (SFN) (1.77 µg/mL) were used as the positive control. Statistically significant differences compared to untreated cells were calculated: p value < 0.05 (*); <0.01 (**); <0.001 (***).

Figure 8

HPLC-MS chromatographic profiles of chromatographic fractions obtained from A. sulcata with and without symbiont homogenates. The most intense peaks of (A) 20% fraction W and W/O and (B) 40% W and W/O were highlighted and tentatively identified.