Improved innate immune responses by Frondanol A5, a sea cucumber extract, prevent intestinal tumorigenesis

Abstract

Sea cucumbers are a source of antibacterial, anti-inflammatory, and anticancer compounds. We show that sea cucumber extract Frondanol A5 is capable of enhancing innate immune responses and inhibiting intestinal tumors in APC(Min/+) mice. APC(Min/+) mice were fed semi-purified diets containing 0, 250, or 500 ppm FrondanolA5 for 14 weeks before we assessed intestinal tumor inhibition. Dietary Frondanol A5 suppressed small intestinal polyp sizes and formation up to 30% (P < 0.02) in males and up to 50% (P < 0.01) in females. Importantly, 250 and 500 ppm Frondanol A5 diet suppressed colon tumor multiplicities by 65% (P < 0.007) and 75% (P < 0.0001), compared with untreated male APC(Min/+) mice. In female APC(Min/+) mice, both dose levels of Frondanol A5 suppressed colon tumor multiplicities up to 80% (P < 0.0001). Isolated peritoneal macrophages from treated mice showed increased phagocytosis efficiency (control 24% vs. treated 50%; P < 0.01) and an increase in GILT mRNA expression, indicating increased innate immune responses by these cells in treated animals. Similarly, we observed an increase in GILT expression in treated tumors, compared with untreated tumors. Furthermore, an increase in G-CSF cytokine, a decrease in inflammatory cytokines and marker 5-LOX, its regulator FLAP, proliferation (PCNA), and angiogenesis (VEGF) markers were observed in treatment groups. These data suggest that Frondanol A5 decreased inflammatory angiogenic molecules and increased GILT expression and macrophage phagocytosis. These decreases may have improved the innate immune systems of the treated mice, thus aiding in inhibition of intestinal tumor formation. These results suggest that Frondanol A5 exhibits significant chemopreventive potential against intestinal tumorigenesis.

©2015 American Association for Cancer Research.

Figures

Figure 1

(A) Active ingredients in sea cucumber extract Frondanol® A5. Fucosylated Chondroitin Sulphate, 12-Methyltretradecanoic acid, and Frondoside A (B) Experimental design for Frondanol® A5 dietary feeding in APCMin/+ mice from 6 weeks of age until termination. Ain76A was the control diet. The experimental groups (diet + drug) and total number of animals per group are listed. (C) Male APCMin/+ mice were exposed to two different doses of Frondanol® A5. Changes in body weights were recorded every week from 6 weeks of age to 20 weeks of age (n=10, Mean±SEM). (D) Female APCMin/+ mice were exposed to two different doses of Frondanol® A5. Changes in body weights were recorded every week from 6 weeks of age to 20 weeks of age (n=10; Mean±SEM). We observed statistically significant differences in body weight gains with both genders.

Figure 2

Efficacy of Frondanol® A5 on intestinal tumors of male and female APCMin/+ mice. (A) Inhibition of total small intestinal polyps (SIPs) formation in APCMin/+ male mice by 250 and 500 ppm of Frondanol® A5. Data are means ± SE of ten animals. Control and treated groups are significantly different from one another (P<.02). (B) Average number of colon tumors per mouse in control and treated APCMin/+ male mice. We observed a significant inhibition of colon tumors with both low-dose (P<.0078) and high-dose (P<.005) Frondanol® A5. (C) Inhibition of total small intestinal polyps (SIPs) formation in APCMin/+ female mice by 250 and 500 ppm of Frondanol® A5. Control and treated groups are significantly different from one another (Low dose P<.01, high dose P<.007) per treatment group. (D) Average number of colon tumors per mouse in control and treated APCMin/+ female mice. We observed a significant inhibition of colon tumors with both low-dose (P<.0001) and high-dose (P<.0003) Frondanol® A5. (E) The size distribution of polyps in small intestines of male mice. (F) The size distribution of polyps in small intestines of female mice. For (E) and (F) * p<0.05; ** p<0.001 versus the unfed control; Data are means ± SE of ten animals per treatment group.

Figure 3

Effect of 500 ppm Frondanol® A on inflammatory cytokines in serum samples from treated (High Dose) and untreated APCMin/+ male (A) and female (B) mice (N=3 / group), as analyzed by ELISA. All assayed cytokines, except G-CSF and GM-CSF, were decreased by Frondanol® A5. IL-2, IL-4, and TNF-α were lowest/undetectable in sera from treated animals.

Figure 4

Modulatory effects of Frondanol® A5 on 5-LOX, FLAP, and VEGF mRNA expression in CTs (A) and SI polyps (B) of treated (High Dose) and untreated APCMin/+ male mice. We observed significantly decreased expression of 5-LOX, FLAP, and VEGF mRNA (VEGF188 and VEGF164) variants after Frondanol® A5 treatment (500 ppm). Decreased 5-LOX and VEGF protein expression in the nucleus of Frondanol® A5-treated (500 ppm) intestinal tumor cells. Serial paraffin sections of colon tumors (C) and intestinal (D) from APCMin/+ were subjected to immunohistochemical analysis using an anti–5-LOX and anti-VEGF polyclonal antibody. A marked decreased accumulation of 5-LOX and VEGF is clear in the cytoplasm and nucleus of tumor cells in treated animals, compared with tumor cells from control animals.

Figure 4

Modulatory effects of Frondanol® A5 on 5-LOX, FLAP, and VEGF mRNA expression in CTs (A) and SI polyps (B) of treated (High Dose) and untreated APCMin/+ male mice. We observed significantly decreased expression of 5-LOX, FLAP, and VEGF mRNA (VEGF188 and VEGF164) variants after Frondanol® A5 treatment (500 ppm). Decreased 5-LOX and VEGF protein expression in the nucleus of Frondanol® A5-treated (500 ppm) intestinal tumor cells. Serial paraffin sections of colon tumors (C) and intestinal (D) from APCMin/+ were subjected to immunohistochemical analysis using an anti–5-LOX and anti-VEGF polyclonal antibody. A marked decreased accumulation of 5-LOX and VEGF is clear in the cytoplasm and nucleus of tumor cells in treated animals, compared with tumor cells from control animals.

Figure 4

Modulatory effects of Frondanol® A5 on 5-LOX, FLAP, and VEGF mRNA expression in CTs (A) and SI polyps (B) of treated (High Dose) and untreated APCMin/+ male mice. We observed significantly decreased expression of 5-LOX, FLAP, and VEGF mRNA (VEGF188 and VEGF164) variants after Frondanol® A5 treatment (500 ppm). Decreased 5-LOX and VEGF protein expression in the nucleus of Frondanol® A5-treated (500 ppm) intestinal tumor cells. Serial paraffin sections of colon tumors (C) and intestinal (D) from APCMin/+ were subjected to immunohistochemical analysis using an anti–5-LOX and anti-VEGF polyclonal antibody. A marked decreased accumulation of 5-LOX and VEGF is clear in the cytoplasm and nucleus of tumor cells in treated animals, compared with tumor cells from control animals.

Figure 5

In vitro phagocytosis of yeast cells by peritoneal macrophages from untreated and treated mice (500 ppm). Continuous feeding of Frondanol® A5 at the tumor initiation stage led to an increase in phagocytosis by peritoneal macrophages in vivo. Mice were exposed to Frondanol® A5 diet or control diet for 14 weeks and then necropsied. Peritoneal macrophages were harvested, plated on glass slides in petri plates, and examined by microscopy to evaluate the presence of internalized dead yeast cells. These peritoneal macrophages were evaluated for GILT mRNA expression. (A) Microscopic picture of adhered macrophages isolated from mice after 18 h incubation. (B) mRNA expression of GILT in control and Frondanol® A5-treated microphages. (C) mRNA expression of GILT and IFN-γ in control and Frondanol® A5-treated CTs and SIPs. (D) Phagocytosis was determined after staining with tannic acid to differentiate adherent yeast cells from those internalized by macrophages. Microscopic image representing the control and Frondanol® A5-treated macrophages after phagocytosis with yeast cells. The magnified image displaying the internalized yeast cells in Frondanol® A5-treated (500 ppm) sample. Arrow indicates internalized yeast. (E) The percentages of phagocytes in control and treated macrophage populations were determined. (F) The percentages of phagocytes with one or two yeast cells in control and treated macrophage populations. (G) The percentages of phagocytes with three or more yeast cells in control and treated macrophage populations. The same slides for analyzing the percentages of phagocytosis are used for these measurements. Quantification of phagocytosis; * P<0.05; n=3, separate experiments with three mice per experiment, over 100 macrophages per mouse.

Figure 6

IHF staining for Annexin 5 was done for apoptotic cells in intestinal tumors from APCMin/+ mice fed control diet or treated with Frondanol® A5 (500 ppm). (A) A significant induction of apoptosis (green staining) was observed in polyps of treated mice compared with untreated mice. A significant difference was observed in apoptotic index between Frondanol-treated (500 ppm) and control group polyps. (B) A significant induction of apoptosis (green staining) was observed in CTs of treated mice compared with untreated mice. A significant difference was observed in apoptotic index between Frondanol-treated (500 ppm) and control group CTs.