Assessment of Potential Anticancer Activity of Brown Seaweed Compounds Using Zebrafish Phenotypic Assay

Abstract

Despite the extensive work on anticancer drug discovery, the number of potent lead compounds that enter the preclinical and clinical trials thus far is still low due to the poor selectivity and understanding in pharmacodynamics. In view of the homology between zebrafish embryogenesis and carcinogenesis in human, zebrafish embryos can be used in the screening platform to elucidate the molecular targets of potential anticancer compounds. In the present study, the possible targets modulating the potential anticancer effects of selected brown seaweed-derived compounds (ie alginate, fucoidan, phloroglucinol, fucosterol, and fucoxanthin) were examined. Teratogenic effects induced by the compounds were observed after 72 hours post-fertilization. Fucoidan, phloroglucinol, and fucosterol were observed to significantly reduce the pigmentation of the zebrafish in a dose-dependent manner at low concentrations (fucoidan, <60 µg/mL; phloroglucinol, <10 µg/mL; fucosterol, <3 µg/mL). On the other hand, embryos treated with fucoxanthin at 200 µg/mL and 300 µg/mL exhibited either phenotypes of curved trunk or bent tail. Further validation work using dual antiplatelet therapy (DAPT) and dorsomorphin as positive controls suggest that fucoxanthin might target the Notch and bone morphogenetic protein (BMP) pathways, respectively. Findings from this exploratory study henceforth have demonstrated the utility of zebrafish embryo to accelerate the discovery of potential compounds for targeted anticancer therapy.

Research based on natural products has led to the discovery of lead compounds for treatment of disease including cancer, inflammation, diabetes, cardiovascular, and infectious diseases. ¹ However, the identification, isolation, and characterization of potential bioactive compounds from the biodiversity are costly, tedious, and time-consuming. Zebrafish (Danio rerio) is emerging as an ideal model for preclinical drug screening of bioactive substances ² due to its genetical similarity (84%) with human disease genes. ³ For instance, embryogenesis in zebrafish is similar with carcinogenesis in human, ⁴ thus the phenotypic defects observed in the zebrafish embryos post compounds treatment indicate the compounds are potent to inhibit the carcinogenesis pathway in human.

Seaweeds are an important source of various bioactive substances, ranging from low-molecular-weight to high-molecular-weight compounds that have been shown to exhibit many medicinal properties including antioxidant, antimicrobial, anti-inflammatory, and anticancer. ^5,6 Notably, the anticancer effect of fucoidan and fucoxanthin, both derived from brown seaweeds, have been extensively studied ⁷ but detailed mechanism of action is still lacking. In the present study, alginate, fucoidan, phloroglucinol, fucosterol, and fucoxanthin—representing some of the major chemical components of brown seaweeds ⁸ —were selected and evaluated for their ability to induce phenotypic defects in zebrafish embryos.

The acute toxicity of the seaweed compounds were determined based on the positive outcome in any of 3 apical observation including coagulation of the embryo, lack of somite formation and nondetachment of the tail in every 24 hours, and the LC₅₀ values were then calculated. All compounds were nontoxic to the embryos at the tested concentrations, except for alginate, with LC₅₀ of 245 µg/mL after 24 hours treatment period.

Ten embryos were treated with each concentration of the compounds. The phenotypic defects of the embryos at 3 days post-fertilization were recorded. Alginate at a concentration of 1 µg/mL induced hooked tail in 10% of the embryo. No other phenotypic defects were observed in embryos treated with alginate at higher concentrations (data not shown). Thus, data indicate that alginate, within the tested concentrations, has no teratogenicity effect on zebrafish embryos.

Fucoidan (Figure 1), phloroglucinol (Figure 2) and fucosterol (Figure 3) induced depigmentation of the embryos as quantified from the yolk sac region, but the effects were observed at different concentrations for each compound. Fucoidan reduced embryo pigmentation only at concentrations lower than 100 µg/mL (Figure 1a). The depigmentation effect of fucoidan diminished at a concentration of 100 µg/mL and above (Figure 1b). The depigmentation in embryos by phloroglucinol is significant (P < 0.005) only at low concentrations 1 μg/mL and 3 μg/mL (Figure 2a) and the pigmentation gradually back to a stage that was comparable to the negative control at concentrations more than 30 μg/mL (Figure 2b).

Figure 1

Fucoidan reduced the pigmentation of zebrafish embryos. (a) Zebrafish embryos treated at different concentrations of fucoidan. Picture shown is a representative of all embryos with same defect. Magnification: 4× (b) Percentage of mean pigmentation was quantified in the yolk sac region using Image-J. Data shown as mean ± SEM of 3 independent experiments. *P < 0.05, **P < 0.005 versus untreated control (0 µg/mL).

Figure 2

Phloroglucinol reduced the pigmentation of zebrafish embryos at concentration lower than 10 µg/mL. (a) Pigmentation of the zebrafish embryo at different concentrations in the yolk sac region. Picture shown is a representative of all embryos with same defect. Magnification: 4× (b) Percentage of mean pigmentation was quantified using Image-J. Data shown as mean ± SEM of 3 independent experiments. *P < 0.05, **p < 0.005 versus untreated control (0 µg/mL).

Figure 3

Fucosterol reduced pigmentation of zebrafish embryo at low concentrations. (a) Zebrafish embryo at different concentrations of fucosterol. Picture shown is a representative of all embryos with the same defect. Magnification 4×. (b) Percentage of mean pigmentation was quantified using Image-J. Data shown as mean ± SEM of 3 independent experiments. *P < 0.05, **P < 0.005 versus untreated control (0 µg/mL).

In contrast, fucosterol induced hypopigmentation as observed in the yolk sac region (Figure 3a). The decrease in pigmentation in 10 embryos was quantified and found to be significant only at low concentrations (0.1, 3 µg/mL), but not in concentrations higher than 3 µg/mL (Figure 3b).

Fucoxanthin did not induce lethality in the embryos up to the tested concentration of 300 µg/mL. Unlike other compounds, fucoxanthin did not induce reduction in pigmentation on the embryos at all concentrations (Figure 4a and b). However, at high concentrations (200 and 300 µg/mL), fucoxanthin induced phenotypic defects of either bent tail or curved trunk (Figure 4c) that were consistent in all embryos.

Figure 4

Phenotype defects induced by fucoxanthin. (a) Zebrafish embryo at different concentrations. Picture shown is a representative of all embryos. (b) Percentage of mean pigmentation from the yolk sac region was quantified using Image-J. Data shown as mean ± SEM of 3 independent experiments. (c) Phenotypic defects of embryos after 48 hours post-treatment, 200 µg/mL: bent tail (40%) or curved trunk (60%), 300 µg/mL: bent tail (30%) or curved trunk (60%). Abbreviation: BT: bent tail, CT: curved trunk. Magnification: 4×.

The curve trunk and bent tail defects observed in fucoxanthin-treated embryos might indicate the possibility of fucoxanthin interfering in the Notch and BMP pathway, respectively. To validate our observation, the embryos were treated with DAPT, a common Notch inhibitor ^9,10 and dorsomorphine, a BMP inhibitor. ¹¹ Fucoxanthin-treated embryos showed similar defects to those treated with DAPT (Figure 5a) and dorsomorphin (Figure 5b). Our data, when taken together, suggest that fucoxanthin may target both the Notch and BMP pathways.

Figure 5

Notch inhibitor (DAPT) and BMP inhibitor (dorsomorphin) induced curve trunk and bent tail, respectively. Zebrafish embryos were treated with DAPT at 100 µg/mL and dorsomorphin at 30 µg/mL. Picture shown is a representative of all 10 embryos with same defects. Magnification: 4×. Abbreviation: CT: Curved trunk; BT: Bent tail.

The phenotypic changes observed in the embryos post treatment with compounds could provide a clue on the implicated cancer pathways due to the homology between embryogenesis of zebrafish and carcinogenesis in human. ⁴ Alginate showed negative and insignificant in phenotypic defect at all tested concentrations after 48 hours of treatment (data not shown). This could be partially attributed to its structure in which high molecular weight compounds are known to be impermeable to the embryos’ membrane.

Fucoidan, phloroglucinol, and fucosterol showed significant reduction in pigmentation in the embryos in a dose-dependent manner at low concentrations, and such diminished at high concentrations. The observation might be due to solubility issues that typically arise when lipophilic compounds are dissolved in organic solvents.

The large colloidal structures, possibly formed when the compounds were prepared at high concentrations, prevented the compounds from diffusing across the cell membrane of embryos thus losing its activity at high concentrations. ¹² Tyrosine kinase pathway was reported to regulate melanocyte pigmentation and development. ^13,14 In zebrafish phenotypic assay, the depigmentation was reported to be associated with interference of the tyrosine kinase downstream effector in carcinogenesis, including PI3K, mTOR, CDK, Flk1, and more. ¹⁵ In cellular and molecular model, the reduced in pigmentation by fucoidan, phloroglucinol, and fucosterol were reported to inhibit cancer cell growth through the PI3K/Akt/mTOR pathway. ^{16 -18} In addition, seaweed extracts have been studied to inhibit melanin synthesis, induced hypopigmentation in B16 melanoma cell line and zebrafish embryos. ^19,20 The inhibition was also shown to be attributed to the inhibition of the tyrosine kinase activity. ^19,20 Hence, we postulated that the compounds could affect the aforementioned pathways based on related findings. ^{16 -20} As both cell culture and zebrafish phenotypic assay yielded the same outcome in many previous studies, the zebrafish phenotypic assay is henceforth a fast and accurate model for target-specific, anticancer drug discovery.

Unlike other compounds, fucoxanthin did not interfere with the mTOR pathway at low concentrations as reported by a previous study. ^19,21 This study revealed that fucoxanthin at high concentrations (200 and 300 µg/mL) target the Notch or BMP pathways, as confirmed by positive control DAPT and dorsomorphin, respectively. In oncology, the inhibition of Notch and BMP pathways were associated with the suppression of metastasis of breast cancers. ^22,23

The high concentrations of brown seaweed compounds used in this study did not cause mortality in zebrafish embryos, proven that these compounds are nontoxic. Hence, the use of zebrafish in this study provides information on toxicity and phenotype defects specifically, which are difficult to be observed in other models of cancer study.

As a conclusion, 4 brown seaweed compounds, namely fucoidan, phloroglucinol, fucosterol, and fucoxanthin used in this study were shown to induce phenotypic changes during zebrafish embryogenesis that can be possibly used to infer some of the pathways targeted by the compounds. Nonetheless, further work using cellular and molecular approaches are warranted to confirm the precise molecular targets of fucoxanthin in both pathways.

Experimental

Compounds

Brown seaweed compounds (alginate, fucoidan from Fucus vesiculosus, fucosterol, phloroglucinol, and fucoxanthin) with purity of >93% (high-performance liquid chromatography) were purchased from Sigma Aldrich (USA). These compounds were dissolved in dimethyl sulphoxide at 50 mg/mL and stored at −20°C.

Zebrafish Husbandry and Phenotypic Assay

The zebrafish were purchased from Danio Assay Laboratory Sdn. Bhd, Malaysia. The fish were maintained at 25°C with a fixed 11 hours photoperiod per day. Healthy adult male and female zebrafish were chosen and placed into mating chamber with transparent divider overnight for 13 hours in dark. After 13 hours, the divider was removed for mating. The eggs were collected after mating. Unfertilized eggs were discarded and fertilized embryos that were uniformly divided at 16 cell stage were selected and placed into the 1 × E3 embryo media (5.0 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl, and 0.33 mM MgSO₄) for treatment with different brown seaweed compounds at different concentrations for 48-96 hours. Ten embryos were placed in each well of 24-well plates for treatment. ³ The concentrations used were based on the OECD guideline. ²⁴

Lethality and Phenotypic Defects

Embryos were evaluated for lethal and phenotypic defects at 72 hours post-fertilization using a CZM4 stereo microscope (Labomed) under magnification of 4×. Embryos stages were monitored, ²⁵ and the lethal or phenotypic defects were recorded. ²⁴ When more than 50% of tested embryos showed the effects, the results were considered as endpoints.

Statistical Analysis

All data were compared and analyzed using the Mann-Whitney t-test (IBM SPSS version 22). P < 0.05 is considered significant.