sea cucumber as anticancer agents and its...

85Copyright © 2014, Squalen BMFPB, ISSN 2089-5690

Squalen Bulletin of Marine & Fisheries Postharvest & Biotechnology, 9 (2), 2014, 85-96

SEA CUCUMBER AS ANTICANCER AGENTS AND ITS DEVELOPMENTFOR FUNCTIONAL FOOD PRODUCTS

Teripang sebagai Agen Antikanker dan Pengembangannya untuk

Produk Pangan Fungsional

Sherly Ridhowati1, 2 *, Fransiska Zakaria2, Dahrul Syah2, and Ekowati Chasanah3

1Sriwijaya University, Jl. Palembang Indralaya Km 32, Ogan Ilir, South Sumatera2Faculty of Agricultural Technology, Department of Food Science, Bogor Agriculture University, Jl. Raya Darmaga, Bogor

3Research and Development Center for Marine and Fisheries Product Processing and Biotechnology, Jl. KS Tubun, Petamburan

VI, Slipi, Central Jakarta, Indonesia, *Correspondence Author: [email protected]

ABSTRACT

Indonesia is popularly known as sea cucumber (teripang) exporter in the form of dried teripang.Commonly known as beche-de-mer or gamat, sea cucumber has long been used as medicineand food by Asian and Middle East people. Recent study has shown that sea cucumbers containactive compounds that show potential health benefits and other biological properties such asantibacterial and antifungal products, anticoagulants, antihypertensives, immuno modulation,inhibitor of osteoclastogenesis. It was reported that sea cucumber posses aphrodisiacs, potentiallyimprove immunity, anticancer and anticoagulation. Sea cucumber is also rich in collagen as acomponent of connective tissue which can further be converted into smaller molecule and act asbioactive substances. This review presents the potential of sea cucumber as a functional foodespecially to prevent cancer and strategy to develop sea cucumber-based functional food byenzymatic hydrolysis and in vivo study.

Keywords: anticancer agent, sea cucumber, enzymatic, in vivo

ABSTRAK

Indonesia populer sebagai negara eksportir teripang dalam bentuk teripang kering yangpada umumnya dikenal sebagai beche-de-mer atau gamat, teripang telah lama digunakansebagai obat dan makanan oleh masyarakat di Asia dan Timur Tengah. Studi terbaru menunjukkanbahwa teripang mengandung senyawa aktif yang berpotensi memiliki manfaat kesehatan danfungsi biologis lainnya seperti produk antibakteri dan antijamur, antikoagulan, antihipertensi,modulasi sistem imun, inhibitor osteoklastogenesis. Dilaporkan pula bahwa teripang berpotensisebagai aprodisiaka, dan berpotensi meningkatkan imunitas, antikanker dan antikoagulan.Teripang juga kaya akan kolagen sebagai komponen jaringan pengikat yang lebih lanjut dapatdiubah menjadi molekul lebih kecil yang dapat berfungsi sebagai komponen bioaktif. Tulisan inimenyajikan potensi teripang sebagai pangan fungsional terutama untuk pencegahan kankerserta strategi penelitian untuk mengembangkan pangan fungsional berbasis teripang melaluihidrolisis teripang secara enzimatik dan studi in vivo.

Kata Kunci: agen antikanker, teripang, enzimatik, in vivo

Article history:Received: 10 April 2014; Revised: 18 Juli 2014; Accepted: 25 Juli 2014

Available online at website: www.bbp4b.litbang.kkp.go.id/squalen-bulletin

1. Introduction

In the last few decades, functional foods offer anew and practical approach to achieve optimal healththrough natural products with physiological benefitsreducing the risk of various chronic diseases. Ashwell(2001) has defined functional food as foods intendedto be consumed as part of normal diet and containsbiological active components that potentially enhancehealth or reduce risk of disease. Most of available

functional foods can be used as therapeutic agents(Bordbar et al., 2011), either indirectly or directly fromnatural sources. Sea cucumber is one of marine-derived natural sources. According to the updatedtrade and catch data available, Asia and Pacific arethe two top sea cucumbers producer regions. It isestimated that the combined harvestable catches ofsea cucumber for the Asia-Pacific regions are in therange of 20,000 to 40,000 ton per year. The medicinalproperties of these animals are ascribed to the

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Copyright © 2014, Squalen BMFPB, ISSN 2089-5690

presence of functional components with promisingmultiple biological activities (Bordbar et al., 2011).

Sea cucumbers belong to the phylumEchinodermata (thorn-skinned animals). They areusually soft-bodied echinoderms comprised of adiverse group of flexible, elongated, worm-like, withleathery skin and a supple body which looks likecucumber. Sea cucumbers tend to live in the sea floorof the deep sea (Tuwo, 2004). Sea cucumbers,commonly known as beche-de-mer, or gamat, haslong been used as medicine and food by Asian andMiddle East people. However, edible sea cucumberspecies with high economic value have only beenfound from the family Aspidochirotae genus Holothuria,Muellaria, and Stichopus. Moreover, the genusHolothuria, Muellaria, and Stichopus are only found inIndonesian seas (Tuwo, 2004).

Information on the potential health benefits and otherbiological properties of the sea cucumber, has beengrowing rapidly in modern biomedical research,especially in the field of neutracetical, such asantibacterial and antifungal products (Aryantina, 2008),anticoagulants (Mulloy et al., 2000), antihypertensives(Zhao et al., 2007), immuno modulation (Gowda etal., 2009) and inhibitor of osteoclastogenesis (Kariyaet al., 2004). There are a series of other bioactive andantiagent substances discovered in sea cucumbers,such as triterpene glycosides, enzymes, amyloses,fatty acids and cytotoxins. Their medical potential useincludes increasing immunity, resisting tumor andanticoagulation, protecting nerve tissue, relieving painand resisting epiphyte as well as contributing toimmunopotentiation, anticancer and anticoagulation.Recently, extract of sea cucumber has been reportedto have the ability to reduce the growth of cancer, thereby opening new perspectives for the development offunctional foods and pharmacology from marineorganisms (Guadalupe et al.,2012).

Sea cucumbers was also reported as aphrodisiacsdue to their bioactive compounds, such as triterpeneglycosides (saponins), chondroi tin sulfate,glycosaminoglycans, polysaccharides sulfate, plantsterols (glycosides and sulfates), phenolics, peptides,collagen, cerbrosida, and lectins (Bordbar et al., 2011).Sea cucumbers are rich in proteins that containessential amino acids, mainly glycine, glutamic acidand arginine. Glycine may stimulate the productionand release of IL-2 and B cell antibody which contributeto the increasing of phagocytosis. Moreover, glycineand glutamic acid play a role as an essentialcomponent to synthesize glutathione that lead tostimulating NK cell activation and proliferation. Argininemay improve immune cells by promoting the activationand proliferation of T cells (Bordbar et al., 2011). Aminoacid components of sea cucumbers have an important

role in the regulation of immune function. Most (70%)of the body wall of sea cucumber protein consists ofcollagen (Saito et al., 2002). Collagen is a componentof connective tissue that can further be converted intogelatin, so that it can be developed as a functionalfood based on the bioactive substances (Bordbar etal., 2011).

2. Anticancer

According to the National Cancer Institute (2012),the colon cancer death ranks third in the world.Whereas the highest incidence rate of lung cancer isfound in men, and for breast cancer, the highestincidence rate is in women. The WHO estimates thatin 2030 there will be increase in cancer patients inIndonesia until seven times (DEPKES, 2010). Cervicalcancer is the first ranks for cancer mortality inIndonesia (DEPKES, 2010), followed with othercancers, such as colon cancer that ranks seventh inIndonesia. Although colon cancer is not the firstleading cause of mortality, most of colon carcinomasare caused by diets, besides various exogenous/endogenous carcinogenic compounds. The coloncancer may ameliorat when risk factors such asgenetic, life-style, and environmental factors aremodified. Diet has been epidemiologically shown tobe closely associated with human colorectal cancer.Chemoprevention of cancer using antimutagens andanticarcinogens present in foods or natural productshas been suggested by various studies, offering themost effective means for preventing human coloncancer (Naveena et al., 2010).

Cancer is a disease caused by DNA damage thatmakes mutations in vital genes involved in controllingcell division. Cell division is a physiological processthat occurs on the tissues. The balance betweenproliferation and programmed cell death are maintainedunder normal circumstances, usually in the form ofapoptosis, cells tightly regulate both processes.Specific mutations in DNA causes cancer throughinterfering with the programming process of celldivision. Carcinogenesis is the process of normal cellsturn into cancer cells (Figure 1). It is characterizedby the development of changes in the cellular andgenetic level, which reprogram the cell to undergouncontrolled division, thus formed a malignant mass(tumor) that spread into distant locations (Guadalupeet al., 2012).

The actual prevalence of cancer may be avoidedthrough diet as an alternative prevention and treatmentof cancer, by consuming natural ingredients (Zakaria,2001) that can be from marine biological resources.Recent research reported that sea cucumbers havebioactive compounds against certain cancers. Tian



et al. (2005) have conducted the in vivo and in vitrostudy on philinopside E compound (PE) of seacucumbers as anti-angiogenic agent. Their researchshowed that the presence of in vitro inhibition by thesea cucumber compound, through proliferation,adhesion, migration, tube formation and apoptosis inhuman umbilical vein endothelial cells (HUVECs) andhuman microvascular endothelial cells (HMECs). Theyassessed through an in vitro trial angiogenesisinhibition potential of the compound using differentassays. Furthermore, they also used in vivo toexamine the PE-inhibition activity on the physiologicalangiogenesis using chorioallantoic membran (CAM)assays. The researchers was using western blottingtechnique to appraise the efficacy of PE on thevascular endothelial growth factor (VEGF) attributingbiosignal in HMECs. The results revealed that PEconsiderably inhibited the proliferation of HMECs andHUVECs, with IC

50of 2.22 ± 0.31 M and 1.98 ± 0.32

M, respectively and induced endothelial cellapoptosis at amounts less than 2 M, showing aconcentration-dependent suppression of cell migrationand cell adhesion as well as tube formation in HUVECsand HMECs. Similarly, in an in vivo CAM assay, PE(5 nM/egg) showed suppression of spontaneousangiogenesis, and exhibited growth inhibitionconsiderably in experimental mouse (sarcoma 180and hepatoma 22) models. The PE takes an importantrole as an efficient anti-angiogenic agent, forsuppressing the active (phosphorylated) forms ofvascular endothelial growth factor receptors involvedin the endothelial cell survival, adhesion, proliferationand migration of the endothelial cells. The PEcompound was able to inhibit the proliferation of

HMECs and HUVECs based on in vivo study (Tian etal., 2005).

Philinopside A, from sea cucumber (Pentactaquadrangulari), was tested against angiogenesis andtumor growth by Tong et al. (2005) both in vitro and invivo models. It possessed dual cytotoxic andantiangiogenic effect that might be attributed to itsinhibitory potential for receptor tyrosine kinases(RTKs). The results indicated that philinopside Asubstantially inhibited the proliferation, migration andtube formation of human microvascular endothelialcells (HMECs). The inhibition depends on the doseapplied, with average IC

50 values of 1.4 ± 0.17, 0.89 ±

0.23 and 0.98 ± 0.19 M, for proliferation, migrationand formation of HMECs. About 2–10 M philinopsideA restrained the formation of new microvessels incultured rat aortas using rat aortas culture assay whichmimics angiogenesis process in vivo.

Philinopside A compound (2–10 nmol/egg)inhibited angiogenesis using chick embryochorioallantoic membrane assay. Furthermore,philinopside A demonstrated strong anti-tumoractivities both in vitro and in vivo. Philinopsidedecrease mouse sarcoma 180 tumor volume bystimulating apoptosis of tumor and tumor-associatedendothelial cells examined using immuno fluorescentanalysis. An examination of the effects of philinopsideA on the angiogenesis-related receptor tyrosinekinases (RTKs) indicated that philinopside A generallyinhibited all tested RTKs, including vascular endothelialgrowth factor (VEGF) receptor, fibroblast growth factor(FGF) receptor-1, platelet-derived growth factor(PDGF) receptor- and epithelial growth factor (EGF)receptor, with IC

50 values ranging from 2.6–4.9 M.

Figure 1. The process of normal cells turn into cancer cells (Picture courtesy of Guadalupe et al.,2012).

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Those results implied that philinopside A can beproposed as a candidate for anti-cancer agent thatpossesses dual cytotoxic and anti-angiogenic effectsbecause of its inhibitory effects on RTKs (Tong et al.,2005)

The results showed that philinopside A significantlyinhibited the proliferation, migration and tube formationof human microvascular endothelial cells (HMECs) ina dose-dependent manner, with average IC

50 values of

1.40±0.17, 0.89±0.23 and 0.98±0.19 M, respectively.Rat aortas culture assay provided a close imitation ofin vivo angiogenesis process and 2–10 M philinopsideA suppressed the formation of new microvessels incultured rat aortas.Philinopside A compound (2–10nmol/egg) inhibited angiogenesis in chick embryochorioallantoic membrane assay. In addition,philinopside A showed strong anti-tumor activities bothin vitro and in vivo. Philinopside reduced mousesarcoma 180 tumor volume by inducing apoptosis oftumor and tumor-associated endothelial cells throughimmuno fluorescent analysis. An examination of theeffects of philinopside A on the angiogenesis-relatedreceptor tyrosine kinases (RTKs) showed thatphilinopside A broadly inhibited all tested RTKs,including vascular endothelial growth factor (VEGF)receptor, fibroblast growth factor (FGF) receptor-1,platelet-derived growth factor (PDGF) receptor-andepithelial growth factor (EGF) receptor, with IC

50 values

ranging from 2.6–4.9 M. Their results suggested thatphilinopside A is a promising anti-cancer agent thatpossesses dual cytotoxic and anti-angiogenic effectsdue to its inhibitory effects on RTKs.

The anticancer activ ity of three tri terpeneglycosides, intercedensides A, B, and C isolated fromthe sea cucumber (Mensamaria intercedens) havebeen evaluated by Zou et al. (2006).Their structureshave been elucidated using spectroscopic analysis

(NMR and ESIMS) and chemical transformations.Intercedensides A (1) and C (3) have a conjugateddouble bond (22Z,24-diene) in the side chain of theaglycon. Intercedenside B (2) has two -D-xylose andtwo sulfate groups in the carbohydrate chain. All threeglycosides showed significant cytotoxicity against 10human tumor cell lines with effective doses 50 (ED

50)

in the range 0.6-4.0 g/ml. Intercedenside A (1)showed significant in vivo antineoplastic activityagainst mouse Lewis lung cancer and mouse S180sarcoma. Intercedensides A-C merit further study aspotential anticancer agent (Zou et al., 2006).

Silchenko et al. (2007) also studied the anticanceractivity of three new triterpeneoligoglycosides, namelyokhotosides B1, B2, and B3, isolated from seacucumber Cucumaria okhotensis, along with theknown compounds frondoside A (4), frondoside A

1,

cucumarioside A2-5, and koreoside A (Figure 2). 2-D

NMR and MS analysis are used to elucidate thestructures of okhotosides B1-3 on the basis ofspectroscopic data established. Compounds 1–3 weremoderately toxic against HeLa tumor cells. FrondosideA (4) compound showed more potent cytotoxicityagainst THP-1 and HeLa tumor cell lines with the IC

50

values of 4.5 and 2.1 µg/ml, respectively. It decreasedboth the AP-1-dependent trascriptional activitiesinduced by UVB, EGF, or TPA in JB6-LucAP-1 cellsand the EGF-induced NF-B-dependent transcriptionalactivity in JB6-LucNF-kB cells at doses of about 1µg/mL. At the same doses, it increased the p53-dependent transcriptional activity in non activated JB6-Lucp53 cells and inhibited the colony formation of JB6P+ Cl 41 cells activated with EGF (INCC

50 = 0.8 µg/

mL). Their result concluded that compounds 1–3 weremoderately toxic against HeLa tumor cells, butFrondoside A showed more cytotoxic effect againstTHP-1 and HeLa tumor cell lines. Similarly, a novel

Figure 2.The structure of compounds frondoside A (4), frondoside A1, cucumarioside A

2-5, and koreoside A

(Silchenko et al., 2007).



triterpenoid, frondoside A, derived from an Atlantic sea-based sea cucumber species namely Cucumariafrondosa has showed effective growth inhibitoryfunction against human pancreas cancer cells. Theproliferation inhibition potential was followed by themagnitude of marked apoptosis. Frondoside A wassupposed to induce apoptosis through mitochondrialand cascade activation pathways.

Zhang et al. (2006a) used the bioassay-guidedfractionation of the active n-BuOH extract of the seacucumber Holothuria fuscocinerea, resulting in theisolation of three new tri terpene glycosides,fuscocinerosides A (1), B (2), and C (3) (Figure 3),along with two known glycosides, pervicoside C (4)and holothurin A (5), as active compounds causingmorphological abnormality of Pyricularia oryzaemycelia. Compounds 1-5 possess the sametetrasaccharide moiety, 3-O-methyl--D-glucopyranosyl-(1-->3 )--D-glucopyranosyl-(1-->4)--D-quinovopyranosyl-(1-->2)-4-O-sodiumsulfato--d-xylopyranosyl, linked to C-3 of holostane triterpeneaglycones that differ in their side chains and 17-substituents. Their structures were elucidated byextensive spectral studies as well as chemicalevidence. All glycosides showed in vitro cytotoxicityagainst two human tumor cell lines (Figure 3). Zhanget al. (2006b) have also isolated three new sulfatedtriterpene glycosides from exract sea cucumber, i.e.,violaceusides I, II, and III (1–3, resp.), as activecompounds causing morphological abnormality ofPyricularia oryzae mycelia. Their structures wereelucidated by spectroscopic methods including 2D-NMR and MS experiments, as well as chemicalevidence. Compounds 1-3 displayed the similarstructural characteristics for the existence of a 16-oxo group in the holostane-type triterpene aglyconewith the C(7)=C(8) bond, except the differences in theside chains and the tetrasaccharide moieties.

Compound 1 has one sulfate group, while the othersare equipped with disulfated glycosides. The entireglycosides demonstrated considerable cytotoxicitiesagainst human gastric cancer MKN-45 and humancolon cancer HCT-116 cells in vitro (Zhang et al.,2006b).

Compounds 1–3 showed the same structuralfeatures, i.e., the presence of a 16-oxo group in theholostane-type triterpene aglycone with the C(7)=C(8)bond, but dif fer in the side chains and thetetrasaccharide moieties. Compound 1 possesses onesulfate group, while 2 and 3 are disulfated glycosides.All the glycosides showed significant in vitrocytotoxicities against human gastric cancer MKN-45and human colon cancer HCT-116 cells (Zhang et al.,2006).

From the same compounds, Zou et al. (2006) havefound six new triterpene glycosides, intercedensidesD”I (1-6) (Figure 4). These compounds were isolatedfrom the whole bodies of the sea cucumber Mensamriaintercedens Lampert that was found in the SouthChina Sea. Their structures were elucidated byextensive spectroscopic analysis (NMR and ESIMS)and chemical methods. lntercedensides D (1), E (2),G (4), and H (5) have a conjugated double bond system(22Z,24-diene) in the aglycon side chain, whileintercedensides F (3) and I (6) have only a singledouble bond (24, 25) in this same chain.lntercedensides D”H (1-5) manifested significantcytotoxicity (ED

50 0.96"5.0g/ml) against 10 human

tumor cell lines, (Figure 4). Two new triterpeneglycosides, hillasides A (1) and B (2), were isolatedfrom the sea cucumber Holothuria hilla Lesson,together with one known glycoside holothuria B (3).Their structures were deduced by extensive spectralanalysis and chemical evidences. The presence ofconjugated double bonds [22E,24-diene] in the

Figure 3.The structure of compounds fuscocinerisides A (1), B(2), and C(3) (Zhang et al., 2006).

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aglycone of 1 is a rare structural feature among seacucumber glycosides. The two glycosides presentedsignificant cytotoxicity against eight human tumourcell lines (A-549, MCF-7, IA9, CAKI-1, PC-3, KB, KB-VIN and HCT-8) with IC

50 in the range of 0.1–3.8 g/ml

(Wu et al., 2011).

In Ogushi’s et al. (2006), sea cucumbers(Stichopus japonicas) were treated with hot water at98 °C for 60 min to prepare extracts that were used toassess their effect on the proliferation and H

2O

2

susceptibility of human colon adenocarcinoma Caco-2 cells. The growth of Caco-2 cells was significantlyinhibited by sea cucumber extracts in a dosedependent manner. High molecular weight seacucumber extract at a concentration of 0.108 mg/mlcould inhibit the cell proliferation. No growth wasobserved at 1.04 mg/ml of the hot water extract after96h incubation. Cell damage by sea cucumber extractwas ev ident abov e 1mg/ml. H

2O

2 showed

concentration-dependent cytotoxity to Caco-2 cells.In addition, co-administration of sea cucumberextracts intensified the H

2O

2 cytotoxity. Ogushi et al

(2006) used phosphatidylserine translocation (APOPercentage Assay kit) for the induction of apoptosis,terminal deoxynucleotide transferase-mediated dUTP-biotin nick-end labeling (TUNEL), and DNAfragmentation as a DNA ladder.

Althunibat et al. (2009) have investigated the effectof aqueous and organic extracts from three seacucumber species (Holothuria leucospilota, Holothuriascabra, Stichopus chloronotus), on the growth of twohuman cancer cells namely A549 (human non-smalllung carcinoma) and C33A (cervical cancer cells) usingMTT assay. Of the extracts tested, only S.chloronotus-derived extract showed antiproliferativeactiv ity against the tested cancer cel l l ines.Conversely, aqueous extract (AE) from S. chloronotusexhibited more toxicity against C33A cells (IC

50 = 10.0

g/ml) than A549. Whereas AE produced from H.leucospilota and H. scabra revealed no notable action

on the growth of the cancer cel ls within theconcentrations limits employed. Sea cucumberextracts produced by the organic solvents inhibitedthe growth of both the cell lines (A549 and C33A) tovarying degrees. The organic extract (OE) from H.scabra species given greater antiproliferative actionagainst A549 and C33A cells with IC

50 values of15.5

g/ml and 3.0 g/ml, respectively. Furthermore. TheOE from S. chloronotus presented more cytotoxicityagainst C33A cells (IC50 = 6.0 g/ml) but little actionagainst A549 cells (IC

50 =21.0 g/ml). Stress oxidative

and degenerative diseases that can be cured with thepresence of the phenols and flavonoids, come fromthe antiproliferative and anticancer of sea cucumberextracts (Bordbar et al., 2011).

Antiproliferative and anticancer functionality of seacucumber extracts might be ascribed to the presenceof considerable amounts of total phenols andflavonoids which were valued as effective antioxidantsto protect from oxidative stress and degenerativediseases including certain cancers. Janakiram et al.(2010) examined the chemopreventive effects offrondanol A, a glycolipid isolated from sea cucumberCucumaria frondosa, against azoxymethane-inducedrat colon carcinogenesis. They used ACF (aberrantcolonic crypt foci) as an efficacy marker to assessthe proliferation expression levels during this study.Besides, the growth-inhibitory and apoptotic effectsof frondanol A over concentration range of 10–120 g/ml using HCT-116 cell line (Janakiram et al., 2010).

Based on the proliferative activity of water extractsof Stichopus variegatus on spinal astrocytes cell lines,Patar et al. (2012) evaluated the dose-dependant effectof Stichopus variegatus water extracts (SVWE) onspinal astrocytes.The extracts were prepared in fourdifferent concentrations of 0.1; 1.0; 5.0 and 10.0 g/ml. The Epidermal Growth Factor (EGF) at 10.0 ng/ml was used as the positive control. The proliferationassay was performed using [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-

Figure 4.The structure of compounds intercedensides D”I (1-6) (Zou et al., 2006).



2H-tetrazolium] (MTS) assay kit. The proliferation wasshowed as percentages of surviving spinal astrocytesafter 72 h treatments. Their study showed that theSVWE has proliferative effect on rat spinal astrocytesin a dose-dependant manner. At the concentration of10 g/ml SVWE has significantly increased the spinalastrocytes proliferation over a period of 72 h (p<0.01).At the concentration of 5 g/ml, the astrocytesproliferation was increased only at 72 h (p<0.05). TheEC

50 of SVWE was 5.18 g/ml. The Malaysian SVWE

(EC50

=5.18 g/ml) showed potential as a growthpromoting agent to promote proliferation of spinalastrocytes at the concentration of 5.0 and 10.0 g/ml(Patar et al. 2012). Patar et al. (2012) revealed thatisolates of sphingoid cerberosides sea cucumber(Stichopus variegatus) also has cytotoxic effectsagainst human colon cancer cell lines. Cerberosidesisolates have characteristics such as a branched alkylchain at C17 to C19 and the double bond of 1 to 3.Sphingoid sea cucumbers exhibited potent cytotoxicactivity against cancer cells (DLD-1, WiDr and Caco-2 cells) that can reduce cancer cell survival, butdependent on the concentration. Compounds weretested whether it triggers morphological changes suchas condensed chromatin fragments of compoundsthat can increase the activity of caspase-3. Sphingoidcan reduce cell viability by causing apoptosis (Pataret al., 2012). This suggests that sea cucumbersphingoid can serve as a functional food componentfor anticancer.

Himaya et al. (2010) demonstrated that the ethylacetate solvent fraction of the sea cucumberStichopus japonicus (SCEA-F) significantly inhibitedthe productions of NO and PGE

2 by inhibiting iNOS

and COX-2 at their protein and gene levels. Theproduction and the gene transcription of pro-inflammatory cytokines were also inhibited. Theresponsible molecular signaling for these inhibitoryactions was found to be through suppression of ERKand p38 MAPK. Their results indicated that SCEA-Finhibits LPS-induced inflammatory response throughblocking of MAPK signaling pathway in murinemacrophages, thus demonstrated its in vitro anti-inflammatory potential. Therefore it could be suggestedthat SCEA-F could be effectively used in functionalfood preparations.

Roginsky et al. (2010) investigated the effects andmechanism of Frondanol-A5P, a polar extract fromCucumaria frondosa, on growth inhibition andapoptosis of S2013 and AsPC-1 human pancreaticcancer cells. The effects of Frondanol-A5P onproliferation, cell cycle, expression of cell cycleproteins and p21waf1, phosphorylation of MAP kinases,annexin V binding, and caspase-3 activation wereexamined. The result showed that Frondanol-A5P

inhibited proliferation and induced G2/M phase cell

cycle arrest in both cell lines with decreasedexpression of cyclin A, cyclin B, and cdc25c.Frondanol-A5P induced phosphorylation of stress-activated protein kinase and Janus kinase (SAPK/JAK) and p38 mitogen-activated protein kinase (MAP)within 5 minutes. Frondanol-A5P markedly increasedthe expression of p21waf1 messenger RNA and proteinat 3 hours in both cell lines. This effect was reducedby the p38 kinase inhibitor, SB203580. Frondanol-A5Pmarkedly increased annexin V binding and activatedcaspase-3.

Li et al. (2009) carried out in vivo study on thepotential of echinoside isolated from sea cucumber.Their study revealed the anticancer effects andmechanisms of action of the tested compound.Anticancer effects of echinoside A were evaluated invitro and in vivo. TUNEL (Terminal deoxynucleotidyltransferase mediated dUTP nick-end-labeling) andDNA fragmentation assays were applied to examineits ability to induce apoptosis. Echinoside A restrainedthe development of tumors in both mouse models andhuman prostate carcinoma xenograft using nudemouse models. There are two special featuresdemonstrated by Echinoside A in the way of restrainingthe noncovalent binding of Top2 to DNA. The first isdone by play a role in competition with DNA for theDNA-binding site of the enzyme, while the otherfeature is conducted mostly by getting involved withTop2-mediated prestrand passage cleavage/religation equilibrium during the poststrand passagephase. These characteristics made Echinoside Adifferent from other common Top2 inhibitors, thereforethis compound stimulated DNA double-strand split ina Top2-dependent way.

Echinoside A restrained the development of tumorsin both mouse models and human prostate carcinomaxenograft using nude mouse models. There are twospecial features demonstrated by Echinoside A in theway of restraining the noncovalent binding of Top2 toDNA. The first is done by play a role in competitionwith DNA for the DNA-binding site of the enzyme, whilethe other feature is conducted mostly by gettinginvolved with Top2-mediated prestrand passagecleavage/religation equilibrium during the poststrandpassage phase. These characteristics madeEchinoside A different from other common Top2inhibitors, therefore this compound stimulated DNAdouble-strand split in a Top2-dependent way.

Echinoside A inhibited the growth of tumors inmouse models and human prostate carcinomaxenografts in nude mouse models. Echinoside Ashowed unique characteristics of inhibiting thenoncovalent binding of Top2 to DNA by competingwith DNA for the DNA-binding domain of the enzyme

92



and of interfering predominantly with the Top2-mediated prestrand passage cleavage/religationequilibrium over with the poststrand passage one.These features distinguish echinoside A from otherknown Top2 inhibitors. As a result, echinoside Ainduced DNA double-strand breaks in a Top2dependent manner.

3. Peptide from Sea Cucumber as Anti Cancer Agent

Peptides with biological activity released duringdigestion or food processing plays an important rolein the regulation of metabolism and modulation. Inthe parent proteins, peptides in protein are still notbe in active form. However, after being released intopeptides, then they become active peptides. Bioactivepeptides usually have a length of 2-20 amino acidresidues. Several studies have reported peptides witha length of more than 20 amino acid residues, knownas the apoptosome and activation of caspase cascadeinitiation (Lang-Hong et al. 2011; Lee et al., 2006).

Protein in the sea cucumber has a complete rangeof amino acids, both essential amino acids and non-essential amino acids, as has recently been reportedby Chen (2006) and Nurjanah (2008 ) that showed thepresence of, almost all essential amino acids excepttryptophan in the sand sea cucumber Holothuriascabra. The differences were only found in the aminoacids phenylalanine, tryptophan, asparagine andglutamine. Chen (2005) reported the absence ofphenylalanine in Holothuria, Actinopyga, andThelenota.

Protein hydrolyzate from marine sources obtainedby enzymatic digestion was an important source ofbioactive peptides and it has been proven to be a goodsource of antioxidant peptides. Purified peptideshowed cytotoxic effects on some cancer cells suchas HeLa, AGS, and DLD- 1, suggesting its potentialdevelopment and application in cancer therapy.Antioxidants are known to be beneficial for humanhealth because it can protect the body from the harmfuleffects of molecules known as ROS (reactive oxygenspecies). ROS can attack membrane lipids, proteinsand DNA. This ROS can be a causative factor in thedevelopment and progression of many different humandiseases such as cancer.The reduction of high ROSmay prevent the occurrence of cancer, because theinhibition of oxidative stress may lead to reducedgenetic changes such as mutations and chromosomalrearrangements that play an important role in theinitiation of carcinogenesis ( Bordbar et al., 2011).

Apoptosis (programmed cell death) is one of themain mechanisms of cell death in response to cancertherapy. Apoptosis is also a naturally occurring process

and evolution of cells that are no longer useful gearedto death. Apoptosis plays a role and fundamentalprocesses in development, physiology, andhomeostasis. Deregulation, loss of pro-apoptoticsignals or no anti-apoptotic signals can cause a varietyof pathological conditions such as cancer initiation,promotion and progression or treatment failure.Apoptosis normally does not trigger inflammatory orimmune response; and therefore apoptosis of cancercould become a way for the treatment of cancer (Lang-Hong et al. 2011). Modulation of apoptotic pathwaysand selective induction of apoptosis by chemicalagents tend to be a promising approach for cancertherapy (Lang-Hong et al. 2011). For example asshown in Figure 5, in mammals, there are two majorsignaling systems that lead to caspase activation,the extrinsic death receptor pathway and the intrinsicmitochondrial pathway (Lang-Hong et al., 2011).

There has been an increasing evidence that theanticancer peptides from the marine sources havecytotoxicity that can trigger apoptosis by targetingmany cellular proteins, and may induce apoptosisprocess by both intracellular and extracellularpathways. In most normal cells, proto-oncogeneencodes a protein that sends signals to the nucleusto stimulate cell division. Signal transduction proteinstakes place in several stages called transductioncascade. This involves a cascade of signalingmolecules to the membrane receptor proteinintermediates that carry signals into the cytoplasmand in the nucleus of the cell transcription factorsthat activate genes for cell division. At every stage ofthe factors or proteins will enable the next phase(Hartwell, 2007; Robbins & Kumar, 2007).

Wang et al. (2010) made the gelatin from the bodywall of sea cucumber (Stichopus japonicus)hydrolyzed with flavourzyme. Low-molecular-weightgelatine hydrolyzate (LMW-GH) of 700-1700 Da havebeen produced using ultrafiltration membranebioreactor system. Chemiluminescence analysisrevealed that the LMW-GH capture free radicals, high-concentration-dependent, IC

50 values for superoxide

and hydroxyl radicals were 442 and 285 mg/ml,respectively. LMW-GH inhibited melanin synthesis andtyrosinase activity in B16 cells. Furthermore, LMW-GH primarily increased intracellular glutathione(GSH), which can suppress melanogenesis. LMW-GH antioxidation activity, holds the potential of beingused as a valuable ingredient in function food,cosmetics and pharmaceuticals or nutraceuticals.

Research of Perez-Vega et al. (2013) revealed thatthe Isostichopus badionotus’s flour was hydrolysedwith the new simulated in vitro gastrointestinaldigestion with digestive enzymes; pepsin and apepsin–Corolase PP-mixture. It has capability as



ACE-inhibitors, radical scavenging activity, ironreduction capacity and cytotoxic effects againstcolorectal cancer cells HT-29. The hydrolysed productwere fractions containing peptides <3000 Da capableas ACE-inhibitors, an effect augmented with combinedaction of gastric (pepsin) and intestinal enzymes (IC

50

=0.038 ± 0.004 mg/ml). Depending on the hydrolysismethod, low and higher molecular weight peptidesfrom sea cucumber have cytotoxic capacity againstcolorectal HT-29 cells due to their antioxidant activity..

Hydrolyzate contains a multi functional peptide thatis resistant to digestive enzymes, greatly increasingthe probability of meeting the diverse biologicalfunctions. Sea cucumbers Isostichopus badionotusalso found to have high concentrations of several aminoacids (eg Glysin, Arginine and Alanine), which mayplay an important role in the physiological effects suchas reducing serum levels of total cholesterol. Peptidespresent in protein hydrolysates and have biologicalactivity depending on molecular weight and amino acidsequence (Guadalupe et al., 2012). Sea cucumbershydrolysates and ultrafiltered fractions are potentialingredients for functional food.

Protein of sea cucumber meat contains 70%collagen and 37% amino acid (Saito et al. 2002; Pataret al. 2012). Hydrolysis of protein is the method usedto obtain peptides and amino acids from protein foodsthat have better biological activity such as antioxidant,antihypertensive, antimicrobial and antiproliferativethan the parent material, i.e protein. This hydrolysisprocess breaks peptide bonds and produce smallerpeptides or free amino acids. Enzymatic hydrolysis

of protein is a commonly used method. Someproteolytic enzymes used to obtain the hydrolyzateinclude, alcalase, trypsin, pepsin, chymotrypsin,pancreatin, pepsin, and thermolysin. Several studieshave shown that enzymatic hydrolysis likely increasedthe antioxidant activity of hydrolysates produced byincreasing radical scavenging activity. Bioactivepeptides showingpotential antioxidant and anticanceractiv it ies as well as immunostimulatory andantiproliferative effects (Jun et al., 2004; Picot et al.,2006) are found in marine protein hydrolyzates andcollagen fibers,which can serve as a proteinsupplement in food. Antioxidants are potential activecompounds that are suitable for the prevention andtreatment of diseases associated with active oxygenspecies, especially cancer (Bordbar et al., 2011). Notonly peptide but also collagen can be used as acancer preventive agent in the gastrointestinal tract(Lee et al., 2006; Lang-Hong et al., 2011).

4. Strategy Development of Functional Food Based on Sea Cucumber

It is very important to develop strategies forexploiting the potential of sea cucumbers as functionalfood with anticancer activity. Sea cucumbers are richin bioactive compounds, especially peptides andcollagen; and therefore further studies, especially invivo experiments, are very important to understandtheir physiology effect in humans and mechanism asanti colon cancers. Xiukun et al. (2010) reported thatin in vivo situation, apoptosis acts to eliminatepotentially deleterious cells without causing such

Figure 5. Apoptosis induction pathways of marine-derived anticancer peptides. Picture courtesy of Lang-Hong et al. (2011).

94



adverse effects such as inflammatory response andensuing scar formation.

Enzymatic hydrolysis of food proteins isconsidered as an efficient way to recover potentbioactive peptides, since several peptides obtainedby this process have different bioactivities and thesemay represent a potential approach to anticancer drugs(Guadelupe et al., 2012). The diverse range of naturalenvironments where the sea cucumber live incombination with the efficient hydrolysis methodapplied will contribute to the discovery of a wide varietyof sea cucumber-derived natural products.Furthermore, this offers an exciting opportunity to findnovel peptides from this organisms, since the studyof marine animal-derived peptides is still in its infancy.Compared with the peptides found from other sources,marine-derived peptides are reported to be morediverse (Chen et al., 2006). Many cyclic peptides ordepsipeptides have been found in marine organismssuch as sea cucumber. These marine peptides seemto be very useful and promising for biomedicalresearch. There is no doubt that the diversity of marinepeptides, in term of chemical structure and mode ofaction,may provide a rich source for designing veryspecific and potent new pharmaceuticals compoundsagainst a wide variety of diseases (Chen et al., 2006).Another potential exploitation of sea cucumber isdeveloping functional food products, such asingredients that are needed for the substitution orfortification of food.

Based on the description above, future researchshould be directed towards enzymatic hydrolysis ofsea cucumber protein and in vivo study. Flour fromthe sea cucumber Stichopus variegatu could behydrolyzed using gastrointestinal protease to obtainseveral peptides. In vivo study could subsequentlybe conducted to investigate the enzymatic processin gastrointestinal tract. The expected result couldprovide insight into the anticancer effect andmechanism of action of the resulting peptides. It wasreported by WHO (2012) that diets have beenepidemiologically shown to be closely associated withhuman colorectal cancer.

The sea cucumber Stichopus variegatus issuggested to be a good source of functional food,because some of the most prevalent marinecomponents used in food, such as glycosides, aminoacids, collagen, gelatin, and peptides, can be obtainedfrom this invertebrate. Sea cucumber is normallyprocessed as flour that can be applied as foodingredients or supplements, cereal-based product(e.g.breads, breakfast cereal and cake), flavor binding,snack foods and gelatin product. The approach couldinclude delivery of the protected bioactive ingredientto their target site and release under certain trigger

factor, such as enzymes, pH, salts, temperature(Chen et al., 2006). Mechanical and thermal processcommonly applied for food processing are size-classification, heating and evaporation using vacuum-drying, flour-milling, and mixing (Chen et al., 2006).The temperature used should be set up under 70 oCto prevent ingredient from denaturation, oxidation andMaillard reaction during processing and storage. It isnecessary to investigate (Chen et al., 2006). Theformulation of each ingredient, the sensory, and theunderstanding of the relation between bioactiveingredient release to meet specific needs of foodapplications.

5. Conclusions

Finding functional food products represents one ofthe major current challenges for pharmacology andmedicine. Therefore, extensive research efforts havebeen focused on identifying new potential bioactivecompounds from natural resources. Development ofproducts based on marine protein hydrolysates andcollagen still remain largely unexplored. Studies onpeptides obtained from protein hydrolysates andcollagen have revealed that these molecules haveantioxidant and antiproliferative activities, suggestingtheir potential development as anticancer drugs.However, more research should be conducted to betterunderstand their mode of action on the cell cycle arrestor apoptosis of cancer cell lines. Nevertheless, thereis a need for strategies to develop and produce thesecompounds.This could be achieved by utilization ofsea cucumber as functional food products.

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