5 Sustainable Marine Structures | Volume 01 | Issue 02 | July 2019 Distributed under creative commons license 4.0 DOI: https://doi.org/10.36956/sms.v1i2.94 Sustainable Marine Structures http://ojs.nassg.org/index.php/sms ARTICLE Biochemical Composition and Anticancer Effect of Different Seaweed Species (In-vitro and In-vivo Studies) T.H. Ranahewa1* A. D. Premarathna1 R.M.K.K. Wijesundara1 V. Wijewardana1,4 A. P. Jayasooriya2,3 R.P.V.J. Rajapakse1* 1. Department of Veterinary Pathobiology, Faculty of Veterinary Medicine and Animal Science, University of Peradeniya, Peradeniya, Sri Lanka 2. Department of Basic Veterinary Sciences, Faculty of Veterinary Medicine and Animal Science, University of Peradeniya, Peradeniya, Sri Lanka 3. Department of Basic Veterinary Sciences, Faculty of Medical Sciences, University of West Indies, Trinidad and Tobago, West Indies 4. Department of Atomic Energy Agency (IAEA) Vienna, Austria ARTICLE INFO ABSTRACT Article history Received: 1 September 2020 Accepted: 13 October 2020 Published Online: 31 October 2020 Seaweed is an enormous resource comprised with natural bioactive compounds with several therapeutic effects including anticancer activity. In this context, the biochemical composition of seaweed plays a major role. Many biochemical compounds isolated from seaweed, fractions of seaweed and crude extracts has revealed ability of seaweed to fight against several cancer types. In this contrast seaweed extracts inhibit cancer cell growth and proliferation by inducing apoptosis and by inhibiting metastasis activity. In this review, biochemical and anticancer properties of seaweeds are discussed and this will provide the basic information to develop a novel chemotherapeutic drug to challenge the cancers. Keywords: Seaweed Biochemical Bioactive Anticancer Apoptosis *Corresponding Author: T.H. Ranahewa, Department of Veterinary Pathobiology, Faculty of Veterinary Medicine and Animal Science, University of Peradeniya, Peradeniya, Sri Lanka; Email: thakshila77harshi@gmail.com; R.P.V.J. Rajapakse, Department of Veterinary Pathobiology, Faculty of Veterinary Medicine and Animal Science, University of Peradeniya, Peradeniya, Sri Lanka; Email: jayanthar@pdn.ac.lk 6 Sustainable Marine Structures | Volume 01 | Issue 02 | July 2019 Distributed under creative commons license 4.0 1. Introduction Ca n c e r i s a d r e a d f u l d i s e a s e c a u s e s d u e t o uncontrolled cell proliferation and migration [17,26]. Among all human cancer types, lung cancer and colorectal cancer are the major cancers found in male whereas breast cancer, cervical cancer and lung cancer are considered as the common cancers found in female [1,40]. Although chemotherapy and radiotherapy treatments are practiced to cure cancers, the survival rate is still low and many side effects are also reported [23]. Due to the continuing of failure of the availability of effective chemo-preventive agents for cancers, therapeutic agents from biological resources are being experimented to control this malignant disease [8,11,18]. Seaweeds are marine macro algae, found in intertidal and sub-tidal zones and classified based on their pigmentation and chemical composition as Chlorophyta (green algae), Rhodophyta (red algae) and Phaeophyta (brown algae) [6,23]. Laminaria sp., Fucus sp., Ascophyllum sp., Porphyra sp., Ulva sp., Sargassum sp. and Gracilaria sp. are some of the examples for the famous edible seaweed species that comprise with many bioactive compounds coming under the groups of polysaccharides, proteins, lipids, minerals and vitamins [14,1]. Moreover, due the availability of theses bioactive compounds many seaweed species account for promising health promoting effects such as antibacterial, antiviral, anti-inflammatory, antiulcer and anticancer activities [1,13,33,41]. Therefore some secondary metabolites in seaweeds are extracted and incorporated into several products such as foods, medicines and cosmetics [5]. Recently, seaweeds are increasingly being considered as a source with effective anticancer agents that are able to lower the risk of cancer [1,17,26]. In this context, investigation of anticancer drugs with less or no side effects has become an interesting topic in novel research field [37,43]. 2. Biochemical Composition of Seaweeds There are two types of biochemical substances present in seaweeds as high molecular materials and low molecular materials. High molecular materials such as dietary fibres are not absorbed into the human body whereas low molecular materials are absorbed directly [14]. Though seaweeds comprise with several biochemical compounds including carbohydrates, proteins, lipids, vitamins, polyphenols, free amino acids and minerals [7,14,24] the composition varies based on the geographical location, seasonal variation and water temperature [14,24]. S e a w e e d s c o n t a i n 2 0 % - 7 6 % ( d r y w e i g h t ) o f polysaccharides as structural and storage compounds. Cellulose, starch, hemicellulose, fucoidan, alginic acids, Sulfated fucans, alginate and laminarin are the polysaccharides which are mainly present in the seaweeds that provide strength and flexibility to the cell wall [5,13,14]. Out of three seaweed types red seaweeds consist with the highest protein content which accounts for 30%-40% of dry weight whereas brown seaweeds contain 15% and green seaweeds contain 30% of dry weight [34]. Many essential amino acids, such as glycine, alanine, arginine and glutamic acids are present in seaweeds. However, the lipid content of seaweeds is comparatively low (1%-5% of dry weight) and it contains a high proportion of essential fatty acids and poly unsaturated fatty acids such as ω-3 and ω-6 [30]. Vitamin B, C, A and D are the most common vitamins present in the seaweeds. In general, seaweeds accounts for 36% (dry weight) of minerals including, potassium, sodium, magnesium, calcium, sulfur, chlorine, phosphorus, iron, zinc, iodine, copper and some other trace metals [34]. Several studies reported that there are severel types of secondary metabolities with therapeutic effect are present in seaweeds (Table 1) Table 1. Secondary metabolites in seaweeds Family Seaweed Species Secondary Metabolites Reference Pheophyta Fucus vesiculosus Fucus evanescens Ascophyllum nodosum Undaria pinnatifida Sargassum thunbergii Ecklonia cava Fucoidan [3,13,16, 20,32,39,45] Laminaria sp. Laminarin [13,31] Sargassum heterophyllum Laminaria ochotensis Hijikia fusiformis Undaria pinnatifida Ectocarpus siliculosus Fucoxanthin [2, 25, 38] Chlorophyta Nitella Hookeri Ulva fasciata Flavonoids [31] Laurencia glandulifera Dactylone [10] 3. Anticancer Effect of Seaweeds Uncontrolled cell growth and proliferation is the main reason for cancer formation. Chemo-preventive agents extracted from natural resources have got the attention due to the ability to suppress cancer cell formation with lesser or no side effects with compared to available chemotherapeutic agents [20]. Basically, cell death can be caused by any chemotherapeutic agent through the cell DOI: https://doi.org/10.36956/sms.v1i2.94 7 Sustainable Marine Structures | Volume 01 | Issue 02 | July 2019 Distributed under creative commons license 4.0 cycle arrest by targeting necrotic pathway or apoptotic pathway of respective cells [23,26]. Bioactive compounds are able to induce apoptosis in cancer cells, this is one of the key mechanisms in cancer therapy because apoptosis is a kind of programmed cell death which can kill only the targeting cancer cells without causing damage to normal surrounding cells [2,40]. In this context, cell death is evoked through intrinsic mitochondrial pathway or extrinsic death receptor pathway [23,40]. Several studies have proven that seaweed constitute many novel bioactive compounds with anticancer effect and able to induce apoptosis in several types of cancer cells [1,3,9,13,35,37]. Anticancer effect of methanolic extract of Caulerpa r a c e m o s a m e n t i o n e d w a s e x a m i n e d a g a i n s t H L - 60 (Human promyelocytic leukemia) cell line and a remarkable cell growth inhibition was reported by Lakmal, et al., 2014 [21] in dose - dependent manner with compared to normal cell line; vero (Monkey kidney cell line). Apoptotic body formation and DNA damage of treated HL-60 cancer cells were obsereved under fluorescent microscopy and flow cytometric analysis was also showed dose dependent sub-G1 DNA accumulation in HL-60 cell line. Aqueous and methanolic extract Kappaphycus striatum has showed cell growth inhibition activity against HeLa (Cervical adenocarcinoma) cell line and this study explains that the molecular weight, monosaccharide sequence, bond formation and charge of molecules are the characteristics of bioactive compounds present in seaweed extracts which support seaweeds to act as an anticancer agent [22]. Zandi, et al., 2010 [42] has found that the aqueous extract of Sargassum oligocystum shows cytotoxic activity on K562 (Human chronic myelogenous leukemia) and Human Daudi (Burkitt Lymphoma) cell lines in dose dependet manner. Moreover by another study Zandi, et al., 2010 [43] expalins that the aqueous extract of Gracilaria corticata filtered with Whatman paper No.1 filter paper showed more pormising cell growth inhibition results on Jurkat and Molt-4 (Human lymphoblast) cell lines and they emphasis that filtration is the one of the best methods of sterilising the seaweed extarcts since some of the bioactive compounds in seaweeds are heat sensitive. MCF 7 (Human breast adenocarcinoma) cell line treated with aqueous extracts of different seaweed species such as Gracillaria corticata, Ulva fasiata, Chaetomorpha antennina showed growth inhibitory effect and morphological observation such as cell shrinkage and cell shape changes related to apoptotic induction were also observed [4]. M e t h a n o l i c e x t r a c t o f S a rg a s s u m m u t i c u m h a s showed antiproliferative activity by inducing apoptosis in MCF-7 (Human breast adenocarcinoma) cell line. Electron microscopic images with membrane blebbing, apoptotic body formation and microvilli reduction, the morphological changes of treated cells stained with Hoechst 33342 and flow cytometric analysis indicating the accumulation of treated cells at sub-G1 phase provide evidence on inducing apoptosis by Methanolic extract of Sargassum muticum [28]. MCF-7 (Human breast cancer cell line) and HepG2 (Human liver cancer cell line) treated with methanolic extracts of Enteromorpha antenna, Gracilaria corticata and Enteromorpha linza has shown cytotoxic activity with compared to normal Vero cell line (Monkey kidney cell line) [29]. Gomes, et al., 2015 [12] found the Methanolic extracts of Dictyota c i l l i o l a t a a n d D i c t y o t a m e n s t r u a l i s s h o w s d o s e - dependent and time-dependent antiprolifertive effect on HeLa (Human cervical cancer cell line). Nuclear morphology changes such as formation of apoptotic bodies and chromatin condensation in the cells stained with 4, 6-Diamidino-2-phenylindole (DAPI) staining was observed under fluorescent microscope. It is reported that percentage of annexin-positive (annexin V-FITC+/ PI−) cells Flow was increased and it is a sign of early apoptosis process of cells. Further the results highlighted that the methanolic extracts of Dictyota cilliolata inhibits the growth of HeLa cells by blocking the cell cycle at the S phase whereas Dictyota menstrualis shows apoptotic induction without inducing cell cycle arrest. Caspase 3 and Caspase 9 activation empahasis that intrinsic apoptotic induction occurs by the seaweed extracts in the cancer cells. Ethanol extract of Ulva fasciata has showed growth inhibition due to apoptosis induction HCT 116 (Human colon cancer) through mitochondrial pathway by increasing cell at sub-G1 phase and activation of Caspase 3 and Caspase 9 [36]. Fractions of polysaccharides; SP-3-1 and SP-3- 2 extracted from Sargassum pallidum were treated on HepG2 (human hepatoma), A549 (human lung cancer) and MGC-803 (Human gastric cancer) cell lines and evaluated by MTT assay. SP-3 fraction with the highest sulfate content exhibited higher antitumor activity against all tested cancer cell lines. Further, this study states structure, glycosidic linkages and sequence of monosaccharaides effects on the function of the extracted polysaccharides [41]. Anticancer effect of a sterol fraction of Porphyra dentata was evaluated against 4T1 breast cancer cell line and pronounced in dose dependent and time dependent cell proliferation inhibition of was resulted in the treated 4T1 cells and percentage of apoptotic-necrotic cells increased was also increased due to the results of PI and annexin V dual staining [19]. Liu et al. (2016) [23] evaluted the anticancer effect of a DOI: https://doi.org/10.36956/sms.v1i2.94 8 Sustainable Marine Structures | Volume 01 | Issue 02 | July 2019 Distributed under creative commons license 4.0 novel Sulfated Polysaccharides (SPS), extracted from Sargassum integerrimum against A549 (Human lung cancer) cell line. Cell growth inhibition was resulted due to necrosis or apoptosis the treated and control cells were stained using Hoechst 33258 staining and morphological changes were observed. Comparatively, typical apoptotic cell characteristics such as cell shrinkage, cell bubbling, fragment shape nucleus and nuclear shrinkage were observed in the SPS treated cells. Flow cytometric analysis using Annexin V-FITC/PI double staining and JC-1 staining also confirms that the growth inhibition is due to apoptosis induction and further due to loss of mitochondrial membrane potential. Moreover, anti- apoptotic and pro-apoptotic protein markers expression were also tested using western blotting. According to the results, the expression level of P53; tumor suppressor protein was increased and Bcl-2; anti-apoptotic protein expression was down regulated whereas Bax; pro- apoptotic gene expression was decreased. Several studies have found that fucoidan extracted from different seaweed species as natural bioactive compound with anticancer effect (Table 2). Fucoidan is a type of sulphated polysaccharide mainly present in brown seaweeds. There are many studies to evident the induction of apoptosis in human colon cancer cell lines such as HCT-15, WiDr, HCT116 and HT-29 by fucoidan [16,17,20]. Moreover HCT-15 cells treated with fucoidan inhibited the growth of colon cancer cell lines in dose dependent manner through apoptosis induction. The treated cells have been visualized with condensed and fragmented nucleus and Bcl-2 expression was down regulated whereas Bax, Pro-caspase 3 and 9 expressions were up regulated by the fucoidan treated cells there for it proves that this growth inhibition is due to apoptosis induction Table 2. Anticancer activity of different seaweed extracts Extract type Seaweed Species Cancer cell type In-vitro analysis References MTT Assay Morphological chances Cell cycle arrest Gene expression Methanoic Caulerpa racemosa HL-60 (Human promyelocytic leukemia) + + + [21] Sargassum muticum MCF-7 (Human breast adenocarcinoma) + + + [28] Enteromorpha antenna Enteromorpha linza MCF-7 (Human breast cancer cell line) HepG2 (Human liver cancer cell line) + [29] Gracilaria corticata MCF-7 (Human breast cancer cell line) HepG2 (Human liver cancer cell line) + [29] Dictyota cilliolata Dictyota menstrualis HeLa (Human cervical cancer cell line) + + + Caspase 3 Caspase 9 [12] Aqueous Sargassum oligocystum K562 (Human chronic myelogenous leukemia) Daudi (Human Burkitt lymphoma) + [43] Gracilaria corticata Jurkat (Human lymphoblast) Molt-4 (Human lymphoblast) MCF-7 (Human breast adenocarcinoma) + [4,42] Ulva fasiata MCF-7 (Human breast adenocarcinoma) + [4] Chaetomorpha antennina MCF-7 (Human breast adenocarcinoma) + [4] Ethanol Ulva fasciata HCT 116 (Human colon cancer) + + + Caspase 3 Caspase 9 [36] DOI: https://doi.org/10.36956/sms.v1i2.94 9 Sustainable Marine Structures | Volume 01 | Issue 02 | July 2019 Distributed under creative commons license 4.0 in the colon cancer cell lines by fucoidan [16]. Further, HCT116 and HT-29, human colon cancer cell lines treated with fucoidan (5-20 µg/ml concentrations) have also showed remarkable dose dependent and time dependent cell growth inhibition and any significant cytotoxic effect was not reported on FHC (human normal colon epithelial) cell line. This growth inhibition was also due to apoptosis induction via activation of Caspase 3, 7, 8 and 9 [20]. Table 3. Anticancer activity of bioactive compounds isolated from seaweeds Bioactive compound Seaweed species used to extract Fucoidan Cancer cell type Reference Fucoidan Fucus vesiculosus HCT-15 (Human colon carcinoma cells) [16] HT-29 (Human colon adenocarcinoma) HCT116 (Human colon adenocarcinoma) [20] Sargassum sp. Turbinaria sp. Padina sp. WiDr (Human colon adenocarcinoma) MCF-7 (Human breast adenocarcinoma) [17] Sargassum oligocystum Daudi (Burkitt lymphoma cells) K562 (Human chronic myelogenous) [42] Caulerpa racemosa HL-60 (Human promyelocytic leukemia cell line) [21] Laminarin Laminaria digitata HT-29 (Human colon adenocarcinoma) [31] Fucaxanthin Laminaria japonica EJ-1(Human bladder cancers) [44] Undaria pinnatifida HL-60 (Human promyelocytic leukemia cell line) [15] Laminarin is another bioacive compound present in seaweeds examined for anticancer properties. Laminarin extracted from Laminaria digitata has shown anticancer effects against HT-29 colon cancer cell. Laminarin showed an activity against HT-29 cell line in a dose- dependent manner. Accumulation of sub-G1 and G2-M during the flow cytometric analysis and inhibition of phosphorylation and ErbB2 expression reveals that Laminarin induces apoptosis through ErbB signaling pathway [31]. Anticancer effect of fucoxanthin extracted from Laminaria japonica was tested against EJ-1(Human bladder cancers) cancer cell line and observed time and dose dependent cell viability reduction. Cell morpholgical changes observed by contrastand fluorescence microscope and activetion of caspase-3 reveals that this cell viability reduction is due to apoptosis induction [44]. Fucoxanthin extracted from Undaria pinnatifida inhbited HL-60 (Human promyelocytic leukemia) cell line proliferation due to apoptosis induction. The results such as DNA fragmentation resuted from agarose gel electrophoresis a n d s a n d w i c h E L I S A t e s t s p r o v e t h e a c t i v i t y o f fucoxanthin against Leukemia cancer cell line [15]. Further, not only in-vitro studies but also in-vivo studies provide the evidance of anticancer activity of seaweeds. In this contrast, LLC (Lewis Lung Carcinoma) cells were transplanted in C57BL/6 mice and treatments (fucoidan and cyclophosphamide) were started after nine days of transplantation. it is recorded that fucoidan shows and atiancer and antimetastatic activity when the drug is used combined with cyclophosphamide [4]. Abirami & Kowsalya, 2012 [1] investigated anticancer effect of Ulva fasciata using albino mice (in-vivo). DAL (Dalton’s Ascites Lymphoma) cells were transplanted in swiss albino mice and treated with methanolic extract and aqueous extracts of Ulva fasciata once daily for 14 days. The parameters such as hematological parameters, lipid profile, liver function tests, body weight, cancer cell counts were examined and methanolic and aqueous exracts of Ulva fasciata showed in-vivo anticancer effect against DAL cancer type. Further mammary cancer induced female rats were treated with Eucheuma cottonii extract (150 and 300 mg/kg of body weight) for four weeks. 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Anticancer activity of Sargassum oligocystum water extract against human cancer cell lines. European Review for Medical and Pharmaco- logical Sciences, 2010 14: 669-73. [43] Zandi, K. et al. In vitro antitumor activity of Graci- laria corticata (a red alga) against Jurkat and molt-4 human cancer cell lines. African Journal of Biotech- nology, 2010, 9(40): 6787-90. [44] Zhang, Z. et al. Potential chemoprevention effect of dietary fucoxanthin on urinary bladder cancer EJ-1 cell line. Oncology Reports, 2008, 20: 1099-1103. [45] Zhuang, C., Itoh, H., Mizuno, T., Ito, H. Antitumor active fucoidan from the brown seaweed, Umitora- noo (Sargassum thunbergii). 2014, 22(41). DOI: https://doi.org/10.36956/sms.v1i2.94