Accepted Manuscript Title: Anticancer and cancer preventive compounds from edible marine organisms Authors: Marta Correia-da-Silva, Em´ılia Sousa, Madalena M.M. Pinto, Anake Kijjoa PII: DOI: Reference:

S1044-579X(17)30084-6 http://dx.doi.org/doi:10.1016/j.semcancer.2017.03.011 YSCBI 1312

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Seminars in Cancer Biology

Received date: Revised date: Accepted date:

11-1-2017 26-2-2017 31-3-2017

Please cite this article as: Correia-da-Silva Marta, Sousa Em´ılia, Pinto Madalena MM, Kijjoa Anake.Anticancer and cancer preventive compounds from edible marine organisms.Seminars in Cancer Biology http://dx.doi.org/10.1016/j.semcancer.2017.03.011 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Anticancer and cancer preventive compounds from edible marine organisms Marta Correia-da-Silva a,c, Emília Sousa a,c, Madalena M. M. Pinto a,c, Anake Kijjoa b,c* a

Laboratório de Química Orgânica, Departamento de Ciências Químicas e

Farmacêuticas, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal b

Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, Rua de Jorge

Viterbo Ferreira, 228, 4050-313 Porto, Portugal c

Interdisciplinary Centre of Marine and Environmental Research (CIIMAR), Terminal

de Cruzeiros do Porto de Leixões, Av. General Norton de Matos s/n- 4450-208 Matosinhos, Portugal *Corresponding author at: Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal E-mail: [email protected]

ABSTRACT A direct impact of food on health, which demonstrates that dietary habit is one of the most important determinants of chronic diseases such as cancers, has led to an increased interest of the consumers toward natural bioactive compounds as functional ingredients or nutraceuticals. Epidemiological studies revealed that the populations of many Asian countries with high consumption of fish and seafood have low prevalence of particular type of cancers such as lung, breast, colorectal and prostate cancers. This observation has led to extensive investigations of the benefits of compounds present in edible marine organisms such as fish, marine invertebrates (mollusks, echinoderms) and marine algae as cancer chemopreventive agents. Interestingly, many of these marine organisms not only constitute as seafood delicacy but also as ingredients used in folk medicine of some East and Southeast Asian countries. The results of the investigations on extracts and compounds from fish (cods, anchovy, eel and also fish protein hydrolysates), mollusks (mussel, oyster, clams and abalone), as well as from sea 1

cucumbers on the in vivo/in vitro anticancer/antitumor activities can, in part, support the health benefits of these edible marine organisms. Keywords: Antitumor, Cancer chemoprevention, Fish and fish protein hydrolysates, Mollusk, Sea cucumber, Seafood

1. Introduction A large number of studies have demonstrated that dietary habit is one of the most important determinants of chronic diseases such as cardiovascular disease, diabetes, gallstones, neurodegenerative diseases, cataract and several types of cancer. Such an association between dietary habit and disease shows that food has a direct impact on health. Interestingly, it was reported that the predominant forms of cancer, as determined by population and epidemiological studies, are those of the lung and bronchus, breast, colorectal and prostate which are prevalent in the western parts of the world, while their incidence is much lower in Asian countries [1]. In 2007, the World Cancer Research Fund (WCRF) published a systematic review report on food, nutrition, physical activity and prevention of cancer, concluding that food and nutrition have a highly important role in prevention and causation of colorectal cancer and consumption of fish has also been associated with a reduced risk of colorectal cancer [2]. Recently, a great deal of interest has been paid by the consumers toward natural bioactive compounds as functional ingredients or nutraceuticals, especially bioactive compounds derived from marine organisms, which have served as a rich source of health-promoting components [3]. Therefore, this review will focus on the chemopreventive properties of compounds produced by marine organisms which are used as seafood in different parts of the world. Since there are already several recently extensive reviews on the health benefits and chemopreventive effects of compounds derived from marine macro- and microalgae [4 -7], they are not included in this review.

2. Fish and fish products Fish consumption is associated with health benefits due not only to its rich content in proteins of high biological value but also unsaturated essential fatty acids, minerals and vitamins [8]. Moreover, fish proteins are highly sensitive to proteolytic digestion and this enhanced digestibility is due mainly to the absence of strong collagenous fibers 2

and tendon in fish muscle. Additionally, fish proteins are rich in all the essential amino acids, especially methionine and glycine [9]. 2.1.

Cod and cod liver oil

Among the marine captures, polar fishes such as cod, receive special attention since they are not only an excellent low-calorie source of protein but also contain a variety of very important nutrients which are useful in a number of different health conditions. Northern cods, including Atlantic cod (Gadus morhua), Greenland cod (Gadus ogac) and Pacific cod (Gadus macrocephalus), also express abundant Thomsen-Friedenreich Disaccharide (TFD)-containing antifreeze glycoprotein (AFGP) which protects them from freezing. Fish AFGPs are usually composed of tripeptide of alanine-alaninethreonine whose last triad is glycosidically linked to TFD (Galβ1, 3GalNAc). The fact that galectin-3 (gal3), which is one of the 15 members of a β-galactoside-binding lectin family which promotes tumor-endothelial cell adhesion), expressed by the capillary endothelium, participates in docking of cancer cells by specifically interacting with TFD present on their surface, while the circulating gal3 mediates homotypic adhesion of cancer cells by binding to the surface TFD, has led Guha et al. [10] to explore the potential antitumor properties of a TFD-containing natural product that could use as food supplement. For this, they have isolated a TFD-containing glycopeptide of molecular mass 100 kDa (TFD100) from the Pacific cod by affinity chromatography and gel permeation chromatography, and have set up an experiment to verify if this exogenous TFD could block gal3-mediated homotypic aggregation and tumor cellendothelial interaction to prevent metastasis. They have found that TFD100 inhibited both in vitro tube formation of human umbilical vein endothelial cells (HUVECs), by binding to the endogenous gal3, as well as in vivo vascular endothelial growth factor (VEGF)-induced formation of blood vessels in C57BL/6 black mice. Investigation of TFD100 for its capacity to inhibit tumor-endothelial cell interactions revealed that this compound inhibited the binding of HUVECs and androgen-independent prostate cancer cells PC3. Moreover, since human cancer cells are capable of blocking or evading the host-immune response, TFD100 was also investigated for its capacity to protect T-cells from tumor-induced apoptosis. The results showed that TDF100 was also able to inhibit gal3-mediated apoptosis of T-cells.

Since

tumor-endothelial

interaction and

angiogenesis are considered as key steps before cancer metastasis, TDF100 could be considered as a promising antimetastatic agent. Thus, they have suggested that TFD3

containing compound from edible fish like cod could be a promising antimetastatic agent for the treatment of various cancers, including prostate adenocarcinoma. Besides cod, “cod liver oil”, a well-known product of cod industry, which was first used to treat rheumatism and then as a source of vitamins A and D, was considered in recent years as a good source of ω-3 PUFAs. Interestingly, only recently much attention has been paid to the research on the anticancer properties of both ω-3 PUFAs and vitamin D. Although there are several reports on the in vitro effects of vitamin D and ω3 PUFAs on breast, prostate, colorectal and lung cancers, as well as epidemiological studies which suggested an inverse correlation between increasing consumptions of both vitamin D and ω-3 PUFAs and increased cancer risk, there is still no research on the efficacy in cancer prevention and treatment of the combination of these two compounds [11]. 2.2.

Anchovy

Another important capture as food staple is anchovies. Anchovies are small, salt water fish comprising more than 100 different species and are found in large schools throughout the Pacific, Atlantic, and Indian Oceans. However, the most popular place to catch anchovies is the Mediterranean. Anchovies are rich in protein, vitamins and minerals, and for this reason anchovies are largely present in a large part of European, Middle Eastern, and North African cuisines. [12]. On the other hand, anchovy sauce has widely been used as a fermented food in Asia. It is also a rich source of amino acids and peptides [13]. Anchovy sauce has been found to possess some interesting biological activities such as antihypertensive [14] and antioxidant properties [15]. Additionally, Lee et al. [16] have found that the hydrophobic peptide fraction of the anchovy sauce was able to exhibit strong antiproliferative activity against a human lymphoma cell (U937), by induction of apoptosis which was accompanied by an increase of caspase-3 and -8 activities. The active peptide fraction was further purified by silica gel chromatography, TLC and reversed-phase HPLC. Analysis of its molecular weight and amino acid composition by MALDI-MS and Pico-Tag HPLC revealed that the antiproliferative peptide was composed of Ala and Phe, having the estimated molecular weight 440.9 Da [17].

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2.3.

Eel

Another important food fish in East Asia, especially in Japan and Korea, is Anguilla japonica or Japanese eel. They are found in Japan, Korea, China, Taiwan, and Vietnam as well as the northern Philippines, where they are raised in aquaculture ponds. It is well established that the skin of eels is covered with carbohydrate-specific proteins, mucus, and mucopolysaccharides for innate immune reaction [18, 19]. It was reported that the lectin AJL-2 and the AJN-10 peptide, isolated from the skin mucus of the Japanese eel, possessed antibacterial activities against Escherichia coli K 12 and Aeromonas hydrophila, respectively. [20]. Recently, Kwak et al. [21] reported a growth inhibitory activity of the eel skin mucus (ESM), isolated from A. japonica from Korea, on human leukemia K562 cells, by causing apoptosis. The results from the immunoblotting analysis suggested that extracellular signal-regulated kinase 1 and 2 (ERK1/2) and p38 signals could be involved in the ESM mediated-apoptosis. Furthermore, they have also found that treatment with lactose rescued the ESM-mediated decrease in cell viability, thus suggesting that lactose binding lectin-like molecules in ESM may be the apoptosisinducing factors. Therefore, the authors have suggested that the skin mucous of eel could be exploited as a potential new drug candidate for an alternative therapy for human leukemia.

2.4.

Fish protein hydrolysate

It is also important to note that more than 70% of the world fish production, which is mainly derived from marine capture fisheries, has been used for processing [22]. Since the majority of fisheries by-products are used to produce low economic-value products such as fish oil, fish meal, fertilizer, pet food and fish silage [23], much attention has been focused on the identification of bioactive compounds from remaining fish muscle proteins, fish oil, fish bone and internal organs, as high-value products for human consumables. These bioactive compounds, particularly bioactive peptides, have been identified as potential nutraceuticals to improve human health and prevent diseases [24]. One of these by-products is fish protein hydrolysates (FPH) which are obtained by controlled enzymatic hydrolysis. FPH are believed to possess an excellent nutritional value since they have a balanced amino acids composition and high digestibility. However, due to their bitter flavor and strong fishy odor, they are still currently used in animal nutrition [25]. Although, peptides from various fish protein hydrolysates have 5

been shown to possess antihypertensive [26], anticoagulant [27], and antioxidant activities [28], investigations of their anticancer activity is still scarce. Picot et al. [25] have investigated the in vitro antiproliferative activity of eighteen FPH from Atlantic salmon (Salmo salar), Atlantic cod (Gadus morhua), plaice (Pleuronectes

platessa),

blue whiting (Micromesistius poutassou), Atlantic emperor (Lethrinus atlanticus), pollack (Pollachius pollachius), and Portuguese dogfish (Centroscymnus coeloepsis) on two human breast carcinoma cell lines, MCF-7/6 and MDA-MB-231. They have found that the FPH from the three Blue whiting, cod, plaice and one salmon, at 1g/mL, induced significant inhibition of cancer cell growth. Moreover, FPH from the three Blue whiting, which contained low NaCl concentration and 96% peptide, were found to induce growth inhibition of 24.5, 22.3, and 26.3% of MCF-7, and 13.5, 29.8 and 29.2% of MDA-MB-231. The authors have pointed out that these values were in the range of those measured in the presence of the anticancer agents: etoposide, roscovitine and kenpaullone, at 10-6 M concentration. They have found also that the antiproliferative activities of these FPH were dependent on the cell lines used since MDA-MB-231, which is classified as a highly invasive breast cancer cell line, was found to be less sensitive to FPH treatment than MCF-7/6. Moreover, since the FHP from Blue whiting (contained 96% of peptide-nitrogen material) had a peptide content ca. 20% higher than those of other fishes, it was suggested that the antiproliferative activity could be related to the presence of specific peptides exerting a direct cytotoxicity on cancer cells. The authors have concluded that the results they obtained suggested that FPH could represent an interesting source of anticancer peptides or lipids to be explored, however preliminary study of the global fish hydrolysate composition did not allow them to correlate the antiproliferative effect with the presence of any particular compounds, especially peptides of defined molecular weight. Consequently, they have expressed their view that the demonstrated bioactive properties in in vitro screening tests did not give a conclusive prove that fish peptides (or native proteins) exerted the same beneficial effects when consumed by humans. In fact, gastric and intestinal protein digestion would render a great variety of short chain peptides among which only oligopeptides and amino acids can undergo small intestinal uptake into the portal vein and a critical step in this overall uptake process involves a transmembrane protein (peptide transporter 1 or PepT1), located in the brush border that can transport nutrient peptides into the enterocyte [29].

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3. Mollusk 3.1. Mussel Blue mussel (Mytilus edulis) is mostly cultured in Canada, USA, Europe and Africa, and its high nutritive potential as a source of protein, vitamin C, iron, zinc and ω-3 PUFAs is well recognized [30]. Like other successful aquaculture industry, high volume of by-products, consisting of shells, damaged mussels and non-commercialized size mussels, has caused an environmental concern. However, since these by-products are constituted of high quality proteins, they could be an important source of bioactive peptides. Therefore, conversion of blue mussel’s by-products into high value biofunctional peptides could provide a solution for environmental problems caused by aquaculture waste disposal as well as valorization of their low-value products. Based on this perspective, Beaulieu et al. [30] have obtained hydrolysates of whole blue mussels by a commercial Bacillus protease complex with broad specificity to hydrophobic amino acids (Protamex). After fractionation, based on molecular mass, the different fractions were tested for antiproliferative activity against four immortalized cell lines, namely A549 (type II pulmonary epithelial cells), HCT15 (colon carcinoma cells), BT549 (breast carcinoma cells), and PC3 (prostate cancer cells). They have found that only the 50 kDa hydrolysate fraction, enriched in peptides, possessed antiproliferative properties with all tested cell lines, exhibiting high activity towards PC3 and A546 cell lines (causing more than 75% A549 and 80% PC3 mortality at a concentration of 11 μg/mL). The 50 kDa hydrolysate fraction was further purified by cation exchange column into two sub-fractions. However, these two purified sub-fractions exhibited less antiproliferative activities than that of the original 50 kDa hydrolysate against all four cancer cell lines. These results suggested that there was a possible synergy of bioactive molecules such as peptides within the 50 kDa hydrolysate fraction which did not happen in the purified fractions. Therefore, the authors suggested that peptides obtained from the enzymatic hydrolysis of blue mussel by-products can provide health benefits and potential applications as active components for functional foods or neutraceutical and pharmaceutical products. 3.2.

Oyster

Oysters are one type of bivalve mollusks that are eaten by cultures throughout the world. Edible oysters have been a part of the human diet for at least 700 years. Oysters 7

are rich in protein and the average oyster contains close to 2 g of protein. The other components include very high levels of vitamin D, vitamin B12, copper, iron, manganese, and selenium. Oysters also contain high levels of niacin, riboflavin, thiamin, vitamin C, phosphorus, potassium, and sodium. They are also a huge source of beneficial cholesterol, antioxidants, ω-3 PUFAs, and water. These elements of oysters make them an extremely healthy food. Oyster peptides have been reported to possess several interesting biological properties namely antifungal and angiotensin-converting enzyme (ACE) inhibitory activities. It was thought that oyster proteins contain various bioactive sequences which can be converted to different bioactive peptides by enzymatic hydrolysis. Wang et al. [31] have prepared oyster (Crassostrea gigas) hydrolysates, using protease from Bacillus sp. SM98011 in laboratory, pilot- and plant-scales, to test for their antitumor activity against the transplantable sarcoma-S 180 as well as their immunostimulating effects in BALB/c mice. They have found that plant-scale hydrolysate (with 57.9% of peptide content), with the dose of 0.5 and 1 mg/g (body weight) significantly inhibited the growth of the transplanted sarcoma S180 cells with inhibitory rates of 30.6% and 48%, respectively. Contrary to the chemotherapy drug CTX, whose inhibition of the growth of transplanted sarcoma S180 cells was accompanied by a significant decrease of spleen and thymus indices, mice treated with the oyster hydrolysate showed a significant increase of thymus and spleen indices when compared to the control (untreated) group. Therefore, the authors suggested that oyster hydrolysates probably inhibited the growth of these tumor cells by improving the immune function in S180bearing mice. They have also observed that, in contrast to CTX, oyster hydrolysates caused a significant increase in NK cell activity and lymphocyte proliferation. These results suggested that cell-mediated immunity of S180 transfected mice was enhanced by treatment with oyster hydrolysates. Moreover, they have found that, although oyster hydrolysates significantly increased the phagocytic rate in a dose-dependent manner, they had no effect on the phagocytic index. These results indicated that oyster hydrolysates caused an increase in the number of total phagocytes and not phagocyte activation. Therefore, the authors suggested that oyster hydrolysates prepared by enzymatic hydrolysis could have potential application for tumor therapy and as a dietary supplement with immunostimulating effects.

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3.3.

Clam

Clams are bivalve mollusks and can live in both freshwater and marine habitats. There are over 150 different edible species of clams in the world. Clams are foods with high nutritional value since they have low fat, high protein and an above average amount of healthful minerals such as iron, magnesium, selenium, zinc and B vitamins such as niacin. Furthermore, some clams, such as the Asian hard clam (Meretrix meretrix), a marine food and a valuable source of traditional Chinese medicine, contain peptides, proteins, enzymes, polysaccharides, mineral, essential vitamins, essential amino acids, which are considered to be responsible for their nutritional and medicinal functions [32, 33]. Ning et al. [34] extracted and purified the 40 kDa protein from the coleomic fluid of Meretrix meretrix, by ammonium sulphate fractionation, ion exchange and hydrophobic interaction chromatography, and evaluated the in vivo antitumor activity against various cancer cell lines. The purified protein extract was shown to significantly inhibit the in vitro growth of human hepatoma BEL-7402 cells, with an IC50 of 52.2 μg/mL. Additionally, this protein extract also displayed cytotoxicity against human breast cancer MCF-7 and human colon cancer HCT116 cells; however, no effect was observed on the growth of the benign cells such as murine fibroblasts NIH3T3 and human breast epithelial MCF-10A cells, even at the concentration as high as 300 μg/mL. Moreover, morphologic changes of the cell membrane were also visualized by scanning electron microscope. The BEL-7402 cells treated with this protein fraction showed a marked disruption of the cell membrane surface. Additionally, the cell permeability of the BEL-7402 cells, measured by LDH leakage, was found to increase in a dose-dependent manner. Flow cytometry analysis revealed that this protein fraction blocked BEL-7402 cells in G2/M phase, in a dose-dependent manner, through inhibition of tubulin polymerization. DNA ladder assay revealed that the protein fraction also induced weak apoptosis of BEL-7402 cells. These data suggested that the antitumor effect of the protein extract of Meretrix meretrix might be due to the changes in cell membrane permeability and inhibition of tubulin polymerization. Therefore, it could have potential for development of a novel, highly selective and effective anti-cancer drug. Later on, Wang et al. [35] purified a novel polypeptide, called Mere15, from Meretrix meretrix by ammonium sulphate fractionation, ion exchange, gel filtration and reversed phase chromatography. Mere15 was found to exhibit cytotoxicity against several human cancer cell lines. Moreover, Mere15 was also found to significantly 9

suppress the growth of human lung adenocarcinoma A549 xenograft in nude mice. Mechanistic study revealed that the cytotoxicity of Mere15 was associated with G2/M phase arrest followed by apoptosis of A549 cells. Further investigation revealed that Mere15, at the concentration of 12.0 μg/mL, was able to decrease the adhesion of A549 cells by 86.74%. Additionally, Mere15, at the concentration of 15.0 μg/mL, was found to decrease the migration and invasion of A549 by 69.22 and 53.84%, respectively. Furthermore, the enzymes matrix metalloproteinases MMP-2 and MMP-9 of the A549 cells treated with 15.0 μg/mL of Mere15 were down-regulated with the inhibition ratio of 72.00 and 93.24%, and the inhibition rate of mRNA expression of MMP-2 and MMP-9 was 57.54 and 91.22%, respectively. This finding suggested that Mere15 inhibits tumor growth via both pro-apoptotic and antimetastasis pathways. Therefore, this polypeptide could have potential to be developed as a multi-target therapeutic agent for the treatment of human lung cancer [36]. Besides the solid tumor A549, Liu et al. [37] also investigated the effects of Mere15 on the in vitro growth of the human K562 chronic myelogenous leukemia K562 cells. They found that Mere15 inhibited the growth of K562 cells with the IC50 value of 38.2 μg/mL. Moreover, they demonstrated also that the K562 cell death was caused by concentration-dependent induction of apoptosis, with overproduction of reactive oxygen species (ROS) and loss of mitochondrial membrane potential. In contrast to the A549 cells, K562 cells treated with Mere15 showed a G0/G1 phase arrest, in a concentration-dependent manner, suggesting that Mere15 blocks the cell cycle in different phases on different cell lines. Furthermore, it was found that Mere15 caused the disassembly of the microtubule cytoskeleton in K562 cells and inhibited the polymerization of tubulin, by interacting with tubulin and reduce its hydrophobic surface. Therefore, Merer15 could be considered as a broad spectrum anticancer polypeptide which exhibits cytotoxicity not only for solid cancer cells but also for the chronic myelogenous leukemia cells. Consequently, Mere15 could be explored for its therapeutic potential for the treatment of leukemia. Black clam (Chione fluctifraga) is a popular seafood which represents an important income source for the community in the Gulf of California [38]. Garcia-Morales et al. [39] prepared the protein extract of the whole black clam and purified, based on the size of the proteins, by gel filtration chromatography into high-molecular weight and lowmolecular weight fractions. These fractions were tested for in vitro antiproliferative activities against the breast cancer MDA-MB-231, cervical cancer HeLa and human 10

retinal pigment epithelial ARPE-19 cell lines. These fractions were found to inhibit the in vitro growth of MDA-MB-231 and HeLa cells. 3.4.

Abalone

Abalones are extensively cultivated and used as food delicacy in many East and Southeast Asian countries. Not only the protein-rich body part of abalone is used for food consumption, the viscera portion is also used to prepare sashimi or prickle in Korea. Uchida et al. [40] reported that the glycoprotein fraction of the liquid from heattreatment of abalone (Haliotis duiscus hannai) strongly inhibited growth of the tumors of ICR mice or BALB/c mice inoculated subcutaneously with allogeneic sarcoma 180 or syngeneic Meth-A fibrosarcoma; however, this extract did not have antitumor activity in T cell-deficient nude mice (CD-1 nu/nu or BALB/c nu/nu mice). Moreover, the fraction was found to activate the cytostatic activity of peritoneal and alveolar macrophages in vivo. These results suggested that the antitumor activity was not due to a direct toxic effect but to stimulation of a host-mediated response. Lee et al. [41] evaluated the antitumor effect of abalone visceral extract on breast cancer model using 4T1 murine mammary carcinoma cells (that have highly metastatic characteristic at an early stage and mimic the human breast cancer). They have found that oral administration of abalone visceral extract significantly inhibited tumor progression by decreasing the levels of Cox-2, EGF, VEGF and fibroblast growth factor (FGF) in primary tumor as well as in metastatic lesions. Additionally, this extract was also found to potentiate proliferation and cytolytic activity of CD8+ T cells. 4. Echinoderm 4.1.

Sea cucumber

Sea cucumbers, also called trepan, bêche-de-mer, or gamat, belong to the class Holothuroidea (phylum Echinodermata) which has around 1250 described living species worldwide [42]. Sea cucumbers, usually processed into a dried product, are valued as an important seafood delicacy particularly in China, Japan and South Asia [43]. From a nutritional point of view, sea cucumbers are considered to be an ideal tonic due to their profile of high-valued nutrients such as vitamins A, B1, B2, B3, and minerals such as calcium, magnesium, iron and zinc [44].

Moreover, commercially processed sea

cucumbers are also a rich source of crude protein when compared to most of the other seafood. It was claimed that fully dried sea cucumber material, which is sold as 11

nutraceuticals in tabulated or capsulated forms for over-the counter dietary health supplements in the USA and Canada, may have a protein content as high as 83% [44]. Sea cucumbers also contain an interesting combination of valuable amino acids. Another interesting aspect is that a high nutritive value of sea cucumber is supported by essential amino acids/ total amino acids as well as of essential amino acids/ nonessential amino acids ratios of its intestine and respiratory apparatus which are close to the ideal pattern defined by FAO/WHO [45]. Apart from having a high nutritious value, sea cucumbers have long been used in Asiatic folk medicine. Interestingly, the potential medicinal benefits and a myriad of biological activities of sea cucumbers are now being recognized in modern biomedical research which supports the claim that sea cucumber extracts are beneficial for human health and can help reduce the growth of cancer cells [42]. On the other hand, many holothurians, especially the tropical species, are toxic, and it was suggested that triterpene glycosides were responsible for this toxicity [46]. These triterpene glycosides were found to possess a wide range of pharmacological activities, including cytotoxic activity. In the past decade, many researchers have paid more attention to holothurian triterpene glycosides as potential marine anticancer compounds and new insights of the mechanisms underlying their anticancer activities have emerged. The anticancer activities of the holothurian triterpene glycosides have been recently reviewed by Aminin et al. [46] and Janakiram et al. [47]. Therefore, only the in vitro and in vivo anticancer activities against various cancer cell lines together with the molecular mechanisms of frondoside A (6)(Figure 2), a triterpene glycoside isolated from the edible Atlantic sea cucumber Cucumaria frondosa, are discussed and updated in this review. Janakiram et al. [48] reported their study of the chemopreventive efficacy of frondanol A5, an isopropyl alcohol/water extract of the enzymatically hydrolyzed epithelia of the edible North Atlantic sea cucumber (Cucumaria frondosa) which contains, besides fucosylated chondroitin sulfate (1), 12-methyltetradecanoic acid (2), eicosapentaenoic acid (3), canthaxanthin (4)/astaxanthin (5), three triterpene glycosides: frondosides A (6), B (7), and C (8), (Figures 1 and 2), using colonic aberrant crypt foci (ACF) as efficacy marker in azoxymethane-induced colon carcinogenesis in F344 rats. They have found that azoxymethane-treated Weanling male F344 rat of 7weeks of age that were fed with the control diet induced, on average, ca. 146 ACF/colon, and 35 foci/colon containing multiple (four or more) aberrant crypts/focus while rats fed with AIN-76A diet containing frondanol A5 (150 or 450 ppm) showed significant inhibition of total occurrences of ACF/colon (34-55%) and of multicrypt 12

clusters containing three, or four, or more crypts/focus (48-70%). Sulindac (a wellestablished chemopreventive agent and known inhibitor of colon carcinogenesis in animal assays, reduced polyps in patients with familial polyposis, and used for comparison study) was found to be an effective inhibitor of total occurrence of ACF/colon (50%) and of multicrypt clusters containing four or more crypts/focus (56.1%). They have found also that frondanol A5 reduced azoxymethane-induced colonic total ACFs and multicrypt ACFs in a dose-dependent manner. Moreover, treatment with frondanol A5, at 450 ppm concentration, also caused an appreciable increase (66%) in the p21-positive cells and substantial decrease (46%) of proliferating cell nuclear antigen (PCNA)-positive cells in the ACFs compared with the azoxymethane-treated control diet. Furthermore, frondanol A5 also decreased the human colon cancer HCT-116 cell viability, with the IC50 value ca. 65 mμ/mL (after 24 hours), through induction of apoptosis. Western blot analysis revealed a dose- and timedependent phosphorylation of H2AX and caspase-2 in the frondanol 5A-treated HCT116 cells as well as a marked reduction of the protein expression of p21 waf1/Cip1 and cyclin B, in a dose-dependent manner. Moreover, the protein expression of cdc25C was also drastically reduced while there was no phosphorylation and protein expression of histone H3 at Ser-10 (a mitotic marker). Therefore, it was concluded that frondanol A5 mediates G2 arrest and not mitotic arrest. Recently, the same group [49] has investigated the capacity of frondanol A5 in enhancing innate immune responses as well as in inhibiting intestinal tumor in APC

Min/+

mice. They have found that frondanol A5

caused a decrease of small intestinal polyps (SIP) and colon tumors (CT) in both male and female mice. Dietary administration of 250 and 500 ppm of frondanol A5 was found to suppress SIP formation up to 28% in males and up to 50% in females, with a significant decrease in polyp size in both genders. Moreover, the numbers of small, medium, and large polyps on the small intestines were significantly decreased by 19.0% in male and 0% in female, 21% in male and 53% in female, and 19% in male and 81.5% in female, respectively, in the mice fed frondanol A5. Additionally, diets supplemented with 250 and 500 ppm frondanol A5 also suppressed CTs multiplicities by 65% and 75% in male mice while the suppression was up to 80% in female mice for both dose levels, in a dose dependent manner, when compared with control diet. Both doses of frondanol A5 were also found to reduce about 90% of high-grade dysplasia (HGD) as compared with untreated control diet. Furthermore, frondanol A5 was also found to alter circulating inflammatory cytokines. It caused a decrease in the levels of 13

interleukins, IFNγ and TNFα, but a significant increase of G-CSF in male mice. Treatment with frondanol A5, at a dose of 500 ppm, also led to a significant reduction of the protein levels of VEGF (angiogenesis marker) and 5-LOX in the adenomatous intestinal polyps as well as a decrease in the protein expression of proliferating cell nuclear antigen (PCNA) in intestinal tumors.

Additionally, frondanol A5 significantly increased not only peritoneal and intratumoral macrophages but also levels of GILT (gamma-interferon-inducible lysosomal thiol reductase) in isolated peritoneal macrophages and IFNγ protein expression in CTs and SIPs. In vitro phagocytosis assay analysis revealed a significant increase in phagocytes in frondanol A5-treated samples. Therefore, they concluded that frondanol A5 significantly increased macrophage function, which significantly contributed to the hosts’ innate immune responses and this improved immune response was reflected by a decrease in intestinal tumors. Roginsky et al. [50] investigated the effects and mechanism of frondanol-A5P, a polar extract from Cucumaria frondosa, on growth inhibition and apoptosis in S2013 and AsPC-1 human pancreatic cancer cells. They have found that frondanol-A5P inhibited proliferation and induced cell cycle arrest at G2/M phase in both cell lines, with decreased expression of cyclin A, cyclin B, and cdc25c, and increased expression of p21. Additionally, frondanol-A5P also induced phosphorylation of stress-activated protein kinase and Janus kinase (SAPK/JAK) and p38 mitogen-activated protein kinase (MAP) besides markedly increased annexin V binding and activated caspase-3. Li et al. [51] reported that frondoside A (6) was able to inhibit proliferation of AsPC-1 human pancreatic cancer cells, through apoptotic induction, in a concentrationand time-dependent manner. Furthermore, by using western blotting, they found that frondoside A (6) caused an activation of caspases 3, 7, 9, decreased expression of the anti-apoptotic proteins Bcl-2, and Mcl-1 in a time-dependent manner, increased expression of Bax, and increased expression of the cyclin-dependent kinase inhibitor, p21waf1 while expression of p27kip1 was not affected. These findings indicated that frondoside A (6) induced apoptosis in human pancreatic cancer cells through the mitochondrial pathway and activation of the caspase cascade. In vivo study revealed that i. p. administration of frondoside A (6), at a dose of 10 mg/kg/day, markedly inhibited the growth of subcutaneously transplanted ASPC-1 cells in athymic mice after four weeks of treatment (as measured by both tumor volume and tumor weight), and no 14

toxicity was observed during the treatment. In a continuation of the investigation of the antiproliferative effects of frondoside A (6) on pancreatic cancer, Al Shemaili et al. [52] investigated the synergistic effects of frondoside A (6) and gemcitabine on proliferation of the AsPC-1 and S2013 cells. They have found that combinations of frondoside A (6) and gemcitabine produced greater inhibition of proliferation than the calculated additive effects in both AsPC-1 and S2013 cells. The effect of frondoside A (6) and gemcitabine on growth, measured as both tumor volume and tumor weight, was also investigated on AsPC-1 and S2013 pancreatic xenografts in athymic mice. It was found that i.p. administration of frondoside A (6) (100 µg/kg/day) and gemcitabine (20 mg/kg/dose, which is one fifth of the concentration used by the Eli Lilly company) caused significant inhibition of growth of the xenografts. However, at 4 mg/kg/dose, gemcitabine was less effective than at 20 mg/kg/dose. Interestingly, they have found that, at the lowest dose tested, combination of gemcitabine (4 mg/kg/dose) with frondoside A (6) (100 µg/kg/day) was significantly more effective than with either drug alone. Recently, Al Shemali et al. [53] compared the growth inhibitory effects of frondosides A (6), B (7), C (8) (Figure 2) and their aglycones on AsPC-1 and S2-013 cells. They have found that frondoside A (6) exhibited a more potent in vitro effect (IC50 ca. 1 µM) than frondoside B (7) (IC50 ca. 2.5 µM), while frondoside C (8) and the aglycones had no effect. These findings suggested that the additional sulphate group on frondoside B (7) decreases the antiproliferative effect while the addition of two sulphate groups on frondoside C (8) and the removal of the sugar moiety resulted in a complete loss of activity. Moreover, they have found also that, in the mouse xenograft experiment, i.p. administration of frondoside A (6), at the dose of 100 mg/kg/day, had a marked and highly significant inhibitory effect on tumor growth when compared with control. On the contrary, oral administration of frondoside A (6), at the same daily dose, had no significant effect on the tumor size or weight. These findings suggested that frondoside A (6) must not be absorbed intact in any significant quantity from the gut or is rapidly metabolized into inactive compounds by the digestive enzymes in the gut. It is likely that hydrolysis of the glycosidic linkage occurred during the passage through the gut since the aglycones showed no antitumor activity. Pharmacokinetic studies confirmed these results as the plasma levels of frondoside A (6) were approximately 700-800 ng/mL after intravenous (i.v.) administration at a dose of 500 mg/kg, while they were approximately 100-120 ng/mL after i.p. administration and were below the limit of detection of the assay (5 ng/mL) after oral administration. 15

Frondoside A (6) was also investigated for its effects on human breast cancer. Al Marzouqi et al. [54] investigated the impact of frondoside A (6) on human breast cancer cell survival, migration and invasion in vitro, and on tumor growth in nude mice using the human estrogen receptors (ER)-negative MDA-MB-231 breast cancer cells. Frondoside A (6) (0.01-5 µM) was found to decrease cellular viability of both MDAMB-231 (IC50 = 2.5 μM, at 24h) and the non-tumerigenic MCF10-A (control, IC50 > 5 μM, at 24h) in concentration- and time-dependent manner. Frondoside A (6) also markedly induced expression of the p53 protein. Treatment of the MDA-MB-231 cells with frondoside A (6) also caused a significant increase in caspases 3/7 activity which was associated with more than 3-fold increase in caspase 9 activity and 2 fold increase in caspase-8 activity, indicating that frondoside A (6) induced caspase-dependent apoptosis, initiated by the activation of the initiator caspase-9, followed by activation of effector caspase-3/7. Moreover, frondoside A (6) also reduced cellular migration in the MDA-MB-231 cells, in a concentration- and time-dependent manner, as well as impairing the invasion of MDA-MB-231-Luc2 cells in matrigel invasion assay. In vivo, frondoside A (6) (10 mg/kg, i. p., 24 days) was found to strongly decrease the growth of MDA-MB-231 tumor xenografts in athymic mice. Additionally, low concentration of frondoside A (6) (1 μM) was found to enhance the cytotoxic effect of paclitaxel against the MDA-MB-231 cells. Ma et al. [55], in investigating the ability of frondoside A (6) to inhibit metastasis in a syngeneic murine model of metastatic breast cancer, have shown that pretreatment of 66.1 mammary tumor cells with frondoside A (6) at 5 μM/L, prior to injection into mice, reduced lung tumor colony formation by 45%. Moreover, frondoside A (6) also inhibited binding of 3H-PGE2 to both EP4-positive (IC50 ~3.7 μM) and EP2-positive cells (IC50 = 16.5 μM), indicating that frondoside A (6) can antagonize binding of PGE2 to both EP2 and EP4 receptors but with a 4.5-fold higher activity for EP4 receptor. Frondoside A (6), at concentration ranging from 0.1 to 0.5 μM/L, was also found to inhibit the induction of cAMP by PGE2 in a dose-dependent manner which is consistent with an EP antagonist function. Furthermore, frondoside A (6) not only completely blocked EP4- and EP2-mediated cAMP activation (with higher concentration) but also inhibited ERK1/2 activation induced by either ligand in a dosedependent manner, indicating that frondoside A (6) is an effective EP4 antagonist and, in lesser extent, antagonizes EP2 actions. Using the clones of 66.1 expressing shRNA targeting EP4 cells, they have found that the inhibitory effects of frondoside A (6) on 16

cell growth were mediated, in part, through EP4 antagonism. Most importantly, frondoside A (6) also inhibited migration of the mammary tumor cells induced by either PGE2 or PGE1-OH (EP4 agonist), however, frondoside A (6) alone had no effect on basal levels of tumor cell migration in the absence of EP ligand. Simultaneously, Park et al. [56], by using a soft agar clonogenic assay, demonstrated that frondoside A (6), at non-cytotoxic concentrations, had an inhibitory effect on the TPA-induced clonogenic ability of the breast cancer MDA-MB-231 cells. They have found also that treatment of these breast cancer cells with frondoside A (6), at doses above 0.1 μM, not only suppressed TPA-induced MMP-9 activity but also decreased its TPA-stimulated secretion and intracellular expression in a dose-dependent manner. Furthermore, frondoside A (6) also increased the levels of expression of the endogenous tissue inhibitors of metalloproteinases, TIMP-1 and TIMP-2, indicating that TIMP-1 and TIMP-2 are involved in the downregulation of MMP in TPA-stimulated breast cancer cells that have been treated with frondoside A (6). Using cells transiently transfected with AP-1-Luc reporter or kB-Luc reporter plasmids, they have found that the activity of AP-1 and NF-κB promotors was increased with treatment with TPA, but was suppressed by frondoside A (6), in a dose-dependent manner. By using a CHIP-PCR assay, they have found that in vitro binding of NF-κB to the MMP-9 promotor was increased by TPA, but the increase was significantly inhibited by frondoside A (6), indicating that the inhibition of AP-1 and NF-κB signals mediates inhibitory effect of frondoside A (6) on MMP-9 expression. Frondoside A (6) also inhibited the phosphorylation of PI3K/Akt, ERK1/2 and p38 MARK, but not JNK’s which suggested that the antimetastatic effects of frondoside A (6) are due to inhibition of PI3K/Akt, ERK1/2 and p38 MARK signaling pathway. Attoub et al. [57] investigated the effects of frondoside A (6) on lung cancer. They have found that frondoside A (6), at concentrations 0.01-5 μM, was able to cause a decrease in cell viability of the human lung cancer LNM35 (NSCLC), A549, NCIH460-Luc2, human melanoma MDA-MB-435, human mammary adenocarcinoma MCF-7, and human hepatoma HepG2 cells, over 24 hours. They have shown also that frondoside A (6), at concentrations 1 and 2.5 μM, was able to increase caspase 3/7 activity by 2.5- and 10.8 folds in LNM35 cells. Treatment with the lowest dose (0.01 mg/kg/day, i.p.) of frondoside A (6) resulted in reduction of the volume of the LNM35 xenografts by 41% without undesirable effects on animal behavior or body weight. 17

They have showed also that frondoside A (6) was able to significantly reduce microvessel density (measured by CD3 staining) at the periphery of the tumor. Furthermore, chorioallantoic membrane (CAM) assay has also confirmed the angiogenic activity of frondoside A (6) since it not only inhibited basal angiogenesis in a concentration-dependent manner but also completely suppressed formation of new blood vessels induced by bFGF. Frondoside A (6) was also found to inhibit spontaneous angiogenic phenotype in the vascular tube formation assay using matrigel. Using a classic in vitro wound healing model, they have found that frondoside A (6) was able to reduce cellular migration of LNM35 cells in a concentration- and time-dependent manner. In the same manner, frondoside A (6) was also found to impair the invasion of LNM35 cells in matrigel invasion assay. Moreover, they have found that the growth inhibitory activity of frondoside A (6) on LNM35 tumor xenograft was comparable with that produced by cisplatin (46.9%); however, combined treatment of frondoside A (6) with cisplatin resulted in a remarkable potentiation (67.6%) of the cisplatin therapeutic effect. Recently, Dyshlovoy et al. [58] investigated efficacy, toxicity and mechanism of action of frondoside A (6) using human castration-resistant prostate cancer (CRPC) cell lines in vitro and in vivo. They have found that both androgen-independent PC-3 and DU145 cells were equally sensitive to frondoside A (6), while the androgen-dependent LNCaP cells were even more sensitive. They have found also that the values of the IC 50 of the prostate cancer cell lines 22Rv1 and VCaP (both containing androgen receptor splice variant AR-V7, leading to resistance to enzalutamide and abiraterone) were comparable to that of the androgen-dependent LNCaP cells while non-malignant human cells (MRC-9, HEK 293T and HUVEC cells) were not significantly sensitive to the effect of frondoside A (6), when compared to the prostate cancer cell lines. Interestingly, treatment with frondoside A (6) caused a cell cycle arrest at G2/M-phase, whereas DU145 and LNCaP cells did not show any significant changes in cell cycle phase distribution. They have shown also that apoptosis induction by frondoside A (6) was caspase-independent in PC-3 cells but caspase-dependent in DU-145 cells. Western blot analysis of PC-3 and DU145 cells treated with frondoside A (6) showed induction of PARP and caspase-3 cleavage, up-regulation of the pro-apoptotic factor Bax (for DU145 only) and Bad (strong up-regulation in PC-3 cells and moderate up-regulation in DU145 cells), as well as down-regulation of the anti-apoptotic factors survivin and Bcl2. While the level of the anti-apoptotic protein PAK1 was unaltered in both cell lines, the pro-apoptotic protein PTEN was found to be up-regulated in DU145 cells but was 18

not detected in PC-3 cells. Analysis of the microtubule-associated protein 1A/1B-light chain 3 (LC3B-I/II) and cytoplasmic vacuolization led to the conclusion that autophagyrelated processes are involved in the cellular response to frondoside A (6). Furthermore, kinetic

studies targeting

LC3B-II

levels

as a

marker

for

autophagosome

accumulation/degradation revealed that frondoside A (6) is capable of inhibiting the late stages of cytoprotective, pro-survival autophagy in PC-3 cells. Moreover, up-regulation of phosphor-mTOR by frondoside A (6) may also contribute to the inhibition of early stage autophagy. Additionally, global proteome analysis revealed that treatment of the cancer cells with frondoside A (6) caused up-regulation of IL-1β and crkII (proapoptotic function), down-regulation of the active form of cathepsin B (associated with increased invasion and migration) and alteration of keratin 81 (associated with invasion and metastasis). Finally, frondoside A (6) was found to significantly inhibit growth of subcutaneously transplanted tumors of both PC-3 and DU145 cell lines. Histological examinations of the lungs of mice bearing PC-3 tumor cells xenografts and treated with frondoside A (6) showed significant reduction of lung metastasis. Moreover, a threefold reduction of disseminated tumor cells in peripheral blood was observed in frondoside A (6)-treated DU145 bearing mice. Jin et al. [59] compared cytotoxic effects of frondoside A (6) with cucumarioside A2-2 (9), a triterpene glycoside isolated from C. japonica, on leukemia HL-60 cells. They have found that frondoside A (6) exhibited stronger growth inhibitory effect on HL-60 cells than cucumarioside A2-2 (9). Although the cytotoxic effects of both compounds was through induction of apoptosis, the apoptosis induced by frondoside A (6) was caspase-independent while that of cucumarioside A2-2 (9) was caspasedependent. 5. Conclusion The role of seafood as anticancer agents has been well supported by the studies described herein holding great promise in cancer prevention and/or therapy. The anticancer and antimetastatic properties of the glycopeptides, produced by polar fishes such as cods, against prostate cancer cells, the growth inhibitory effects of ω-3 PUFAs against breast, prostate, colorectal and lung cancer cells and the antiproliferative effects of fish protein hydrolysates from Atlantic and polar fishes, support the observation that in a population with higher rates of fish consumption, such as that of Finland and in Swedish fisherman, the incidence and mortality from colorectal cancer are greatly 19

reduced [2]. A number of mollusks such as oyster, clam and abalone are seafood delicacy in many parts of the world. These organisms contain bioactive peptides which have antiproliferative and antimetastatic activities against various cancer cells, including breast, prostate, lung and colon cancers. On the other hand, sea cucumber represents one of the most appreciated seafood delicacy in many Asian countries, contains, besides a myriad of essential amino acids and polyunsaturated fatty acids, a number of triterpene glycosides. Interestingly, frondanol A5, a glycolipid extract of the North Atlantic sea cucumber (Cucumaria frondosa) caused inhibition of colon carcinogenesis in vitro as well as reduction of colon tumors in vivo when administered orally. Similarly, frondoside A, the triterpene glycoside constituent of frondanol A5, was also found to inhibit growth of various cancer cell lines both in vitro and in vivo, when administered intraperitoneally. The fact that frondoside A, when administered orally, did not show the in vivo antitumor activity against pancreatic cancer cells, led to the conclusion that this compound is metabolized and loses its anticancer activity due to hydrolysis of a glycosidic linkage by the digestive enzymes in the gut. Therefore, the anticancer effect of sea cucumber could be due other compounds rather than sulfated triterpene glycosides when consumed as food. However, frondoside A can serve as a lead compound for further development of anticancer drugs. Therefore, the high valuecompounds present in edible marine captures can pave the way for exploring their potential use for functional foods, nutraceuticals or even a new type of anticancer drugs.

Conflict of interest The authors have no competing interest.

Acknowledgements This work was supported by the project INNOVMAR - Innovation and Sustainability in the Management and Exploitation of Marine Resources (reference NORTE-01-0145-FEDER-000035, within Research Line NOVELMAR/INSEAFOOD /ECOSERVICES), supported by North Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). 20

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_

_ R = OSO3

1

2

3

4

5

Fig. 1. Structures of fucosylated chondroitin sulfate (1), 12-methyltetradecanoic acid (2), eicosapentaenoic acid (3), canthaxanthin (4) and astaxanthin (5).

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6

7

8

9

Fig. 2. Structures of frondosides A (6), B (7), C (8) and cucumarioside A2-2 (9).

29