Introduction

This review provides an overview of the evidence that macroalgae-derived natural products provide a potential source for novel anti-cancer drugs. To contextualize their use, a short initial discussion of the issues relating to cancer treatment is provided.

Approaches to cancer treatment

The initial presentation of a tumour is the result of genetic changes which occur in critical genes within a cell that control its normal growth and development. This leads to unregulated proliferation of the cell and its progeny which results, except in the case of most haematological malignancies, in the development of a solid tumour. The increase in tumour size depends on a large number of interacting factors including a reduction in cell death (particularly through the apoptosis pathway), an increase in proliferation and the generation of new blood vessels to provide oxygen and nutrients to support the growing mass (King and Robins 2006). Tumours are classified in relation to their tissue of origin to include squamous cell carcinomas, adenocarcinomas, sarcomas, lymphomas and neuroectodermal cancers; however, even within each group, there is much heterogeneity (Weinberg 2007). Six main characteristics of tumours have been proposed: the ability to proliferate without reliance on external growth signals, insensitivity to anti-growth signals, resistance to apoptosis, limitless replicative potential, the ability to encourage angiogenesis and the ability to metastasise (Hanahan and Weinberg 2011). In many instances, this final characteristic is the one that results in death of the individual since there are very few effective treatments of metastatic cancer. As tumours grow, the genome becomes increasingly unstable with the acquisition of further deleterious mutations that promote malignant progression.

The complexity of tumour pathology makes treatment very challenging. When a patient presents with a single solid tumour, it may be possible to remove it through surgical excision or by sterilization with external beam radiotherapy; however, in many cases, this is not possible. Even when surgery or radiotherapy is used as a first-line treatment, in many patients this approach is not curative. This can be for a number of reasons, e.g. lack of accessibility for complete excision, limitations in the dose of radiation used due to the risk of damage to critical normal structures in the radiation field etc. When surgery or radiotherapy is not possible or limited, then cytotoxic chemotherapy (CCT) is a main treatment option. CCT can be used as a first-line treatment or as an adjunct to other therapeutic approaches. The aim of CCT is to destroy all cancer cells within the body, including those that have spread to distant tissues, i.e. metastasized (Kintzios 2004). Conventional CCT drugs are primarily designed to act on rapidly proliferating cells (King and Robins 2006). Unfortunately, many healthy cells in the body with high rates of proliferation can also be affected, e.g. cells in the bone marrow, intestinal villi and hair follicles. Side effects are consequently moderate to severe and include nausea, anaemia, weakening of the immune system, hair loss, diarrhoea and vomiting. Due to the heterogeneous nature of tumours and their inherent genetic instability, tumours may develop resistance to CCT through selection for resistant subclones. The majority of the most common cancers are therefore not curable with a single CCT drug, and treatment approaches frequently include surgery and/or radiotherapy, where practical, and/or a combination of several drugs (Kintzios 2004).

More recently, research into CCT has focussed on improving the specificity of cytotoxic agents by targeting specific features of the tumour micro-environment including the poorly formed vasculature (Wu and Staton 2012), hypoxic tumour cell sub-populations (McKeown et al. 2007) and infiltrating inflammatory cells (Joyce 2005). In addition, considerable efforts are being made to identify molecular targets specific to tumour cells (Kaufman et al. 2008). However, to date, there have only been a few successes with this approach, and most novel agents still require combination with more conventional cytotoxic drugs and/or surgery/radiotherapy. One approach to identify new CCT agents is to mine the natural environment. This review now focusses on the potential of marine-derived macroalgae as a source of new CCT agents.

Marine natural products as a source of pharmaceuticals

Natural products in medicine

Humans have made use of natural resources as a source of medicinal compounds for thousands of years. In many Asian and African countries, traditional therapies are still widely used as a primary treatment. In addition, many people in developed countries (70 to 80 %) have used some form of alternative or complementary medicine (http://www.who.int/mediacentre/factsheets/2003/fs134/en/). Bioactivity is an inherent characteristic of natural products as they are derived from living materials that use chemicals to interact with other tissues and foreign organisms through binding with specific receptors (Maschek and Baker 2008). This bioactivity can be defined as a physiological response elicited by a molecule binding to a specific ligand; the response may be beneficial or deleterious (Colegate and Molyneux 2008). The most commonly identified bioactive natural products have been shown to elicit anti-oxidant (Jiménez-Escrig et al. 2001), anti-microbial (Hornsey and Hide 1974; Reichelt and Borowitzka 1984; Kubanek et al. 2003), anti-viral (Ponce et al. 2003) or anti-tumour (Usui et al. 1980; this review) activity.

A bioactive substance may be used as a crude extract or as a pure/semi-pure isolate. There are advantages and disadvantages to both approaches. Clearly crude extracts are easier and cheaper to produce; however, they are likely to suffer from seasonal, spatial and morphological variation which will affect the quality of the final product (Colegate and Molyneux 2008). Other compounds will be present in crude extracts, and the effects of these impurities, either synergistic or antagonistic, are rarely known. Isolation of a pure bioactive molecule allows for the dose to be reproducible and structural determination to be carried out which enables elucidation of the mechanism of action (Colegate and Molyneux 2008). A problem with natural product isolation is that yields tend to be low.

It has been reported that over 50 % of prescribed drugs are either natural products or synthetic chemicals that derive from natural products (Newman et al. 2000). This increases to 63 % of drugs used for CCT (Cragg and Newman 2009). Pharmaceuticals from natural products can be derived from many different plant and animal species. A small number of marine-derived compounds have entered clinical trials (Table 1), although only one is derived from seaweed (kahalalide F; see “Protein and peptides”). This is somewhat surprising given the extensive evidence of the potential of marine macroalgae as a source of anti-cancer drugs. This evidence is summarised and discussed below.

Table 1 Anti-cancer agents from marine sources which are currently in clinical development
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Seaweed as a source of pharmaceutical agents

Macroalgae, commonly known as seaweeds, have been consumed in Asia and parts of Europe for centuries. The biggest consumers of seaweed today are China, Japan, Korea, Indonesia, Philippines and Hawaii and to a lesser extent, France, Ireland, Iceland, Norway, Wales and coastal areas of the USA and Canada (Yuan 2008). The benefits of eating seaweed were first identified through epidemiological studies that showed the low prevalence of diseases such as coronary heart disease and diet-related cancers in countries with high seaweed consumption (Kono et al. 2004; Yang et al. 2010b; Teas et al. 2011).

Seaweed is an excellent source of bioactive chemicals. In addition to the primary metabolites required for normal growth, seaweeds produce many secondary metabolites in response to a wide range of fluctuating environmental pressures. These include seasonal changes which influence salinity, temperature and light (Abdala-Díaz et al. 2006), UV radiation exposure (Pavia and Brock 2000) and herbivory (Van Alstyne 1988). Terpenes and polyketides, which are oligomers of the primary metabolites isoprene and acetate, account for most secondary metabolites (Maschek and Baker 2008). It should be noted that there is great diversity in seaweed composition, with many compounds being unique to a group. For example, the pigment fucoxanthin is only present in brown seaweeds, and the carbohydrate ulvan is only present in green seaweeds. There is also natural variability which can occur in the same species of seaweed and even within different parts of the same thallus. Thus, if a potential source of drug is identified, in order to confirm and extend the observations, it is important that all of the above factors are recorded and controlled for, in so far as it is possible. However, this variability is only occasionally referred to in publications although it is of the utmost importance (Stengel et al. 2011).

Over a 22-year period (1960–1982), a large number (16,000) of marine organism-derived samples were screened for anti-tumour activity at the National Cancer Institute (NCI; in the USA). The programme was discontinued as few new chemical leads were discovered. In 1985, a new in vitro 60-cell panel was introduced which allowed easier screening of natural products (Roussis et al. 2004). Despite the diversity of marine-derived products identified currently, just four are approved for use as drugs in Europe (see Table 2). They are also approved in the USA except for trabectedin which is currently in phase III clinical trial (Mayer et al. 2010). It should be noted that synthetic or semi-synthetic routes to these drugs are now known.

Table 2 Drugs in current use originally derived from marine sources
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Seaweed as a source of anti-cancer agents

Anti-cancer agents can be viewed in two ways. When seaweeds are ingested as a nutrient source, epidemiological studies have indicated that they may provide some protection from the development of cancers. This is probably linked to the anti-oxidant properties of their constituent molecules. However, when prepared as a concentrated extract, there is considerable evidence that seaweeds also contain molecules that have cytotoxic properties. This review focusses primarily on the latter. The presence of cytotoxins in macroalgae is not surprising since these molecules will protect against herbivory and encroachment of other seaweeds into their habitat. Recent evidence for this is summarised in Tables 4, 6, 7, 8, 9, 10 and 11. It should be noted that most of these papers have only investigated the effects of crude extracts or semi-pure extracts on cells in vitro. Although this is an essential first step, there are very many issues to be addressed if any of these potential sources is to be translated into a modern pharmaceutical product.

Identification and development of seaweed-derived cytotoxic compounds

The potential of seaweeds as a source of anti-cancer drugs has primarily been investigated using partially purified fractions or crude extracts. In some reports, the exact source of the fractions is unclear. In so far as is possible, these studies have been summarised in Tables 4, 6, 7, 8, 9, 10 and 11 under the type of fraction investigated. Many of the early studies used mouse models to investigate the anti-tumour effects of seaweeds administered either raw or as a crude extract (see Tables 10 and 11). Since the 1980s, research on seaweed extracts has focussed more on isolated fractions, particularly of polysaccharides and also small molecule extracts including terpenes, sterols, macrolides, alkaloids, halogenated phenols and carotenoids (almost exclusively fucoxanthin). Extracted peptides/proteins have also been studied, but to a more limited extent than other chemical groups. More recently, there has been increased interest in the bioactivity of polyphenols.

Polysaccharides

Structure, occurrence and isolation

Polysaccharides are the most widely studied cytotoxic agents derived from macroalgae, and the most common source of these polysaccharides is from brown seaweeds. The sulphated polysaccharide variants such as fucoidans or ascophyllans are based on l-fucose sugars (Berteau and Mulloy 2003). Typical percentages of sulphated polysaccharides found in macroalgae are given in Table 3. In addition, some reports have investigated the unsulphated polysaccharide, alginate. Laminarin has also been studied as a chemically sulphated derivative. Extraction of polysaccharides is normally in aqueous, alkali or acidic medium; for more detailed information, see McHugh (1987), Lahaye and Robic (2007) and Rioux et al. (2007).

Table 3 Typical percentages (on a dry weight basis) of polysaccharides found in macroalgae
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One of the main groups of polysaccharides present in green seaweeds is the sulphated heteropolysaccharides known as ulvans. They are composed mainly of sulphate, rhamnose, xylose and glucuronic acid (Lahaye and Robic 2007). In red seaweeds, the major polysaccharide groups present are sulphated galactans called agar and carrageenans; the latter are more highly sulphated than agars. Porphyran is another type of sulphated polysaccharide found in red seaweeds.

Cytotoxicity

Brown seaweed polysaccharides are the most widely investigated extracts. The majority of these investigations focus on fucoidan whilst other extracts that have been studied include alginate, laminarin or unclassified crude polysaccharide extracts. Most of the extracts classified as ‘brown unidentified and miscellaneous’ (Table 4) are likely to be fucoidan; however, the fractions were not sufficiently characterised or purified for the authors to name the specific polysaccharide. A review focussed on fucoidan has been published recently (Senthilkumar et al. 2013). It should be acknowledged that this lack of clarity on the identification of polysaccharides may in part be due to the difficulty in characterising these complex molecules. Strategies to address this include hydrolysis and high-performance liquid chromatography (HPLC) to discover monosaccharide composition, followed by mass spectrometry (MS) and/or nuclear magnetic resonance (NMR) spectroscopy to further identify substitutions and linkages. The identification of seaweed polysaccharides is further hampered by the structural diversity in polysaccharide type. The polysaccharides in question may have repeating sugar residues in their backbone, but may vary in sugar substitutions, sulphation and molecular weight. Take as an example the review mentioned above, which found that fucoidan extracted from five different seaweed species had fucose contents ranging from 13 to 36 % and degrees of sulphation which varied from 8 to 25 % (Senthilkumar et al. 2013).

Table 4 Polysaccharides from seaweeds with anti-cancer properties
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In the published literature, the analytical identification of macroalgal-derived polysaccharides shown to have activity as CCT agents varies widely. In a few studies, a thorough analysis has been carried out. For example, Thinh et al. (2013) used ion exchange HPLC to study monosaccharide composition and a combination of NMR and MS to elucidate linkage type and sulphation in fucoidan. In others, a general carbohydrate extraction technique and more qualitative analysis has been carried out. In these cases, it is likely that the polysaccharide extract contains impurities and unknown polysaccharide fractions; see, for example, Zhou et al. (2004) who characterised a carrageenan extract by viscosity, total sugars, FTIR and qualitative UV and HPLC. Many other studies report little or no characterisation at all, yet if they are to be progressed it will be essential that further characterisation is done. A review by Jiao et al. (2011) describes several strategies for analysis of polysaccharides from seaweeds. However, considerable time and resources are required to analyse these complex molecules effectively.

Importance of sulphation

Fucoidan is a sulphated polysaccharide. The importance of fucoidan sulphation to cytotoxic activity has been well established. Several studies, both in vitro and in vivo, have compared the activity of native fucoidan with that of fractions which have either been artificially over-sulphated or contain more sulphate by means of the fractionation technique used (Yamamoto et al. 1984a; Koyanagi et al. 2003; Teruya et al. 2007; Croci et al. 2011). For example, an over-sulphated fucoidan fraction, but not a native fraction, had an anti-tumour effect against L-1210 leukaemic cells in mice (Yamamoto et al. 1984a). This differential effect was also reported in studies by Koyanagi et al. (2003) who observed an in vivo enhancement of anti-angiogenic activity in S180 tumours grown in imprinting control region (ICR) mice in a dorsal air sac and anti-tumour effects in LLC and B16 cells implanted in the foot pad of ICR mice. The inhibitory effects were greater for over-sulphated fucoidan, although native fucoidan did have some efficacy. In a study of three fucoidan fractions isolated from Laminaria saccharina (now Saccharina latissima), it was shown that human umbilical vein endothelial cell (HUVEC) tubulogenesis in vitro and matrigel plug vascularisation in vivo was correlated with the level of sulphation, with the greatest inhibition found in the fraction with the highest sulphation (Croci et al. 2011). However, no significant differences were found regarding the degree of sulphation in several fucoidan extracts in a colony formation assay of DLD-1 cells (Thinh et al. 2013).

The importance of sulphation has been shown for other polysaccharides besides fucoidan. Chemically sulphated laminarin, but not sodium laminarin, had anti-heparanase activity in vitro and reduced metastasis in vivo, with a small effect on tumour proliferation and growth (Miao et al. 1999). This anti-heparanase effect is important as it could provide a novel compound with activity against tumour metastasis, a feature of tumours which currently is inhibited by very few drugs. In crude carbohydrate fractions from Sargassum pallidum, more highly sulphated polysaccharides were found to be more cytotoxic to cancer cell lines (Ye et al. 2008). Virtually all the polysaccharides studied in green and red seaweeds are sulphated (see Table 4). When compared to other sulphated polysaccharides, the action of alginate (unsulphated) has been found to be inferior to both fucoidan and ascophyllan (sulphated) (Nakayasu et al. 2009).

Effect on tumour angiogenesis

Tumours require a constant supply of both nutrients and oxygen which can only be achieved if new blood vessels form within the growing mass. Since the vasculature in normal tissues is largely quiescent, targeting angiogenesis in tumours provides a specific target which can inhibit the ability of tumours to grow. This has led to considerable efforts to find compounds (both synthetic and natural) which target tumour vasculature. Several studies have shown that angiogenesis can be inhibited by the sulphated polysaccharides fucoidan and laminarin (see Table 4). No substantial evidence has yet been found to suggest that the unsulphated polysaccharide alginate or the sulphated red and green algal polysaccharides affect angiogenesis.

Although some information can be obtained using tube formation assays in vitro, angiogenesis is a three-dimensional process, and it is mostly studied using in vivo models, primarily involving either chick eggs or various experimental techniques in mice. The chicken embryo chorioallantoic membrane (CAM) assay is commonly used where the substance of interest is implanted in a chick egg membrane and its effect on angiogenesis quantified (Dias et al. 2005). In a study of fucoidan isolated from Sargassum stenophyllum, both the CAM assay and a gelfoam plug assay in mice revealed the fucoidan to have anti-angiogenic activity. As discussed above, Koyanagi et al. (2003) found fucoidans reduced angiogenesis as well as eliciting anti-tumour effects in mouse models. Fucoidan has also been reported to reduce the pro-angiogenic cytokine and vascular endothelial growth factor (VEGF), and this was associated with a significant reduction in angiogenesis and tumour size in 4T1 tumours in vivo (Xue et al. 2012). In a recent report, it has been shown that production of VEGF-A and tube formation is significantly reduced in HUVECs exposed to fucoidan isolated from Undaria pinnatifida (Liu et al. 2012a).

Plasminogen activator inhibitor-1 (PAI-1) is a protein present at elevated concentrations in cancer patients that is produced in endothelial cells and is proposed as a good target for cancer drug development (Andreasen 2007). In a survey of the bioactivity of nine different fucoidans from brown seaweeds, it was reported that some of the fucoidans caused a decrease in PAI-1 release from HUVECs in vitro providing a rationale for their use in developing a targeted anti-cancer drug (Cumashi et al. 2007). As discussed above, decreased HUVEC tubulogenesis was observed on exposure to fucoidans especially those with higher sulphation levels which was also correlated with lower levels of PAI-1 secretion; they also found in vivo effects on angiogenesis (Croci et al. 2011).

A study has shown that a sulphated laminarin (LAM S5) can inhibit angiogenesis in the CAM assay; however, there were issues with toxicity (chick embryo deaths). When they tested LAM S5 against RIF-1 tumours grown in C3H/Km mice, toxicity was also found, with haemorrhagic deaths occurring at higher doses. A synergistic anti-tumour effect was found when mice were treated with LAM S5 in combination with a corticosteroid or a cytotoxic agent (melphalan) (Hoffman et al. 1996). The CAM assay was also used to show that vasculogenesis stimulated by basic fibroblast growth factor (bFGF) could be reduced by a polysaccharide extracted from S. stenophyllum (Dias et al 2008). A recent in vivo study found no differences in the levels of the pro-angiogenic molecules bFGF and VEGF in mice treated with fucoidan, but did find a reduction in tumour weight and volume (hepatocellular carcinoma, Bel-7402). The authors suggested a mechanism unrelated to tumour angiogenesis was responsible for the observed anti-proliferative activity (Zhu et al. 2013).

The effect of molecular weight on polysaccharide bioactivity

In a study of fucoidan extracted from the brown seaweed U. pinnatifida, cytotoxicity was observed against AGS stomach cancer cells; this was significantly greater in the lower molecular weight fucoidan fraction, i.e. <30 kDa compared to >30 kDa fraction (Cho et al. 2011). There is also evidence showing that the red seaweed polysaccharide, carrageenan, has better anti-tumour activity in low molecular weight forms. When S180 or H22 tumours were exposed to extracts of varying molecular weight, the tumours grew more slowly when mice were treated with lower molecular weight or degraded carrageenan (Zhou et al. 2004, 2005, 2006); this was accompanied by immunomodulatory effects. It should be noted that in various studies, degraded carrageenan (also called poligeenan) has been found to induce colitis and ulceration in animal studies (Watt and Marcus 1971; Benard et al. 2010). Concerns have been mostly limited to poligeenan (carrageenan is a high molecular weight polysaccharide). However, there is an ongoing debate regarding the safety of carrageenan (Tobacman 2001, 2002; Carthew 2002; Burges Watson 2008).

Enhancement of immune response

Many studies have shown that polysaccharides can prolong survival in tumour bearing mice; this is often shown to be caused by a reduction in tumour size that is associated with an improvement in the animals’ immune defences. For example, it was reported that fucoidan inhibited the growth of Ehrlich carcinoma cells in mice through an enhancement of the host’s immune function (Itoh et al. 1993). Fucoidan from the sporophyll of U. pinnatifida was found to prolong survival in P-388 tumour-bearing mice. This was associated with a significant enhancement of the activity of natural killer (NK) lymphocytes and increased production of interferon gamma (IFN γ) by T cells (Maruyama et al. 2003). Fucoidan was also found to modulate Th1 and NK cell responses in mice inoculated with leukaemic cells (Maruyama et al 2006). Ale et al. (2011a) also reported that fucoidan enhanced NK cell activity in non-cancerous mice.

The action of an unidentified polysaccharide from Sargassum confusum was examined in S-180 tumours grown in mice. It was found to improve immune function as measured by increased superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) activity and thymocyte and splenocyte production, as well as decreased malondialdehyde (MDA) (Liu and Meng 2004, 2005).

Ascophyllan was found to induce cytokine release (tumour necrosis factor (TNF) and granulocyte colony-stimulating factor) from macrophage-derived RAW264.7 cells (Nakayasu et al. 2009), and a sulphated polysaccharide derived from the brown seaweed Hydroclathrus clathratus was reported to increase TNF-α in mouse serum (Wang et al. 2010).

An in vivo study on λ-carrageenan of various molecular weights found that, in general, NK and lymphocyte cell proliferation in animals implanted with S180 and H22 tumours was higher in animals treated with carrageenan than the negative control (Zhou et al. 2004). The same group also showed that recovery from damage caused by 5-fluorouracil (5-FU) was linked to an improvement in immunocompetence, as measured by an increase in spleen weight and lymphocyte proliferation; this suggested that these extracts may have a role in ameliorating damage caused by standard chemotherapeutic agents such as 5-FU (Zhou et al. 2005, 2006).

Jiao et al. (2009) characterised a sulphated polysaccharide from the green seaweed, Ulva (formerly Enteromorpha intestinalis), with an average molecular weight of 46.8 kDa and a high rhamnose content. Very little in vitro activity was found against S-180 sarcoma cells; however, when the cells were implanted in vivo, there was a reduction in tumour mass, an increase in thymus and spleen mass and an increase in TNF-α production and lymphocyte proliferation. The authors suggested immuno-enhancement rather than direct anti-tumour cytotoxicity as the mechanism of action. Increased levels of the immunomodulators nitric oxide (NO) and prostaglandin E2 (PGE2) were observed when mouse leukaemic cells (RAW 264.7) were treated with a sulphated polysaccharide extracted from Monostroma nitidium; this effect was independent of a direct cytotoxic effect seen against AGS tumour cells (Karnjanapratum and You 2011).

Cell death by apoptosis

Polysaccharides are known to cause cell death primarily via an apoptosis pathway, with the apoptosis-controlling proteins caspases, Bax and Bcl-2 frequently involved. For example, caspase 3 was found to be activated in HS-Sultan (malignant lymphoma) cells treated with fucoidan (Aisa et al. 2005). However, Philchenkov et al. (2007) found that no cytotoxicity occurred in MT-4 and Namalwa lymphoblastoid cells on treatment with 500 μg mL−1 of fucoidan, although pre-treatment with fucoidan prior to etoposide exposure doubled the level of apoptosis in MT-4 cells but not in Namalwa cells. Unlike the previous study (Aisa et al. 2005), they found that apoptosis was not related to caspase-3 activation (Philchenkov et al. 2007). Further evidence of caspase involvement has come from a study of U937 cells; this showed that over-sulphated fucoidan induced apoptosis through a caspase-3 and caspase-7 activation-dependent pathway, whilst the activity of native fucoidan was weak (Teruya et al. 2007). Fucoidan was also found to up-regulate caspases 3 and 9 and Bax (pro-apoptotic proteins) in HCT-15 cells, whilst Bcl-2 and Akt (anti-apoptotic proteins) were reduced (Hyun et al. 2009). In a study of the effects of fucoidan on HT-29 and HCT116 human colon cancer cells, fucoidan was found to cause apoptosis in a dose-dependent manner, whilst having no effect on normal FHC colonocytes. Molecular analysis of the HT29 cells showed that fucoidan increased levels of a number of pro-apoptotic proteins involved in the mitochondrial-mediated pathway of apoptosis; these included cleaved caspases 3, 7, 8 and 9; cleaved poly(ADP-ribose) polymerase (PARP); Bak and truncated Bid. A number of anti-apoptotic effectors were reduced including the X-linked inhibitor of apoptosis protein (XIAP), survivin and Mcl-1. An enhancement of mitochondrial membrane permeability, as well as release of cytochrome c and Smac/Diablo from the mitochondria, was also observed. Further molecular changes, including increased levels of Fas-L, TNF-related apoptosis-inducing ligand (TRAIL) and DR5, implicated the activation of the death receptor-mediated apoptotic pathway (Kim et al. 2010a).

In contrast, no changes in caspases, ERK, p38, p53 or pAKT levels were found to accompany the apoptosis induced by fucoidan in HeLa cells; however, apoptosis inducing factor (AIF) was found in the cytosol, as well as up-regulation of Bax and down-regulation of Bcl-2 (Costa et al. 2011b). In U937 cells, apoptosis was induced by a sulphated polysaccharide from Ecklonia cava; this was associated with an up-regulation of Bax and PARP, activation of caspases 7 and 8 and down-regulation of Bcl-2 (Athukorala et al. 2009).

The sulphated polysaccharide porphyran, isolated from an unidentified red seaweed, was found to induce apoptosis in AGS cancer cells but not in three normal cell lines. This was accompanied by enhanced cleavage of PARP and caspases 3 and 9 and reduced phosphorylation of insulin-like growth factor-1 receptor (IGF-1R) which correlated with Akt down-regulation (Kwon and Nam 2006). In a study of AGS cells exposed to a crude polysaccharide extracted from the green alga Capsosiphon fulvescens, similar results were found; cell proliferation was inhibited and apoptosis induced. This was associated with inhibition of IGF-1R signalling and the PI3K/Akt pathway (Kwon and Nam 2007).

Several other studies have shown that apoptotic tumour cell death is caused by exposure to polysaccharides (Gamal-Eldeen et al. 2009; Nakayasu et al. 2009; Zou et al 2010; Ale et al. 2011a, b; see Table 4). Clearly, although apoptosis is frequently involved in the cytotoxic effect of polysaccharides, there may be different aspects of the apoptotic response involved depending on the experimental cells and the extract composition.

Many studies have clearly shown that polysaccharides, from a wide range of macroalgal sources, have an effect on tumour cell growth both in vitro and in vivo. These compounds act in a variety of ways including direct inhibition of proliferation and activation of apoptosis mediated through a number of different molecular pathways. In addition, they can act indirectly by targeting the blood vessels growing in the tumour and/or via immunomodulation mechanisms that boost the bodies’ natural mechanisms for eradication of tumour cells.

Conclusions

Polysaccharides, and sulphated polysaccharides in particular, have been the most studied groups of macroalgal-based anti-cancer agents. They have shown good potential, and there is much evidence regarding their in vitro and in vivo potential. However, none has entered clinical trials, which may be related to the difficulties encountered when purifying and unambiguously identifying their structure. Since polysaccharides are complex molecules with varied levels of sulphation and sugar moieties, it is likely that even the most pure fractions will contain polysaccharide molecules with a range of structures. In addition, identification of a suitable administration route and the formulating of these relatively large molecular structures will be difficult. With all of the positive evidence for their potential utility, it is perhaps time for these problems to be addressed.

Polyphenols

Structure and occurrence

Polyphenols are secondary metabolites of seaweeds. Their presence and concentrations can be linked to environmental factors (Maschek and Baker 2008). Oxidative stress is toxic in vivo, and the presence of reactive oxygen species (ROS) is normally balanced by anti-oxidant defences. However, when ROS are in excess, they can cause a rise in intracellular Ca2+ and iron, which may damage DNA and is implicated in many age-related degenerative conditions including cancer (Alfadda and Sallam 2012). Polyphenols act as anti-oxidants by preventing the formation of free radicals, binding metal ions and/or improving the body’s own anti-oxidant system (Cox et al. 2010). It should be noted that polyphenols can also have pro-oxidant effects under certain conditions (Perron et al. 2011).

Phlorotannins are oligomers and polymers of phloroglucinol that are found exclusively in brown algae. Phlorotannins bind to metals and proteins, and they have an astringent taste which often makes brown algae unpalatable to humans (Ragan and Glombitza 1986). There is a wide molecular weight range of phlorotannins isolated from Ascophyllum nodosum (0.32–400 kDa), with the majority being about 10 kDa (Ragan and Glombitza 1986). Environmental factors which may affect polyphenol levels in macroalgae include UV radiation exposure (Pavia and Brock 2000), herbivory (Van Alstyne 1988), season (Abdala-Díaz et al. 2006) and geographical location (Tanniou et al 2013). There are fewer studies of polyphenol extracts from green and red as compared to brown seaweeds. Polyphenol levels are lower in the red and green seaweeds and are predominantly a mixture of catechins, gallate catechins and gallic acid (Yoshie et al. 2000; Rodríguez-Bernaldo de Quirós et al. 2010). Environmental factors and the method of isolation have a major effect on the levels of polyphenols extracted; Table 5 summarises the information available.

Table 5 Total polyphenol levels in three different seaweeds (García-Casal et al. 2009)
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Polyphenols are normally quantified according to the Folin–Ciocalteu method calibrated to either gallic acid or phloroglucinol. The results therefore are quoted as gallic acid/phloroglucinol equivalents. The lack of analytical standards for phlorotannins limits the possibility of their characterisation by HPLC. If a standard does not exist, mass spectrometry may prove useful in elucidating the structure. However, HPLC and MS are constrained by the huge molecular weight and polymer type diversity in the compounds although a recent paper has reported some improvements to characterisation methods by HPLC–MS (Steevensz et al. 2012). In general, polyphenols can be extracted using aqueous/solvent systems, sometimes with precipitation of carbohydrates and dialysis to remove salts. More information on extraction methods can be found in Ragan and Glombitza (1986). Bromophenols are polyphenols with one or more bromine substitutions. Marine algae, in particular red and brown seaweeds, are a common source of bromophenolic compounds. Those studied in the literature tend to be smaller phenolic compounds which are well characterised and purified by the authors.

Polyphenols as anti-cancer agents

Seaweed polyphenols are being investigated for their potential utility in two different anti-cancer roles. The first focusses on their ability to directly inhibit cancer cell proliferation and promote cell death. The second is as a cytoprotective agent, which can protect cells against DNA damage and thus reduce the incidence of tumours. To complicate this matter still further, in most studies, the extracts investigated are polyphenol rich with the polyphenol content varying from >1 to >99 % purity. This means that authors in most cases can only tentatively attach a claim of efficacy to the polyphenols in the extract and indeed most acknowledge this. To date, all studies reporting on pure extracts have been carried out on phlorotannins (Hwang et al. 2006; Zhang et al. 2010; Kong et al. 2009; Oh et al. 2011; Li et al. 2011). One example of the cytoprotective effect of polyphenols was shown in a study of extracts from Fucus vesiculosus and Fucus serratus. The Comet Assay was used to measure DNA damage on Caco-2 cells exposed to H2O2; when the cells were exposed to the polyphenol extracts, this reduced the level of DNA damage suggesting cytoprotection, although the effect was small (O’Sullivan et al. 2011).

The direct cytotoxicity of several algal extracts on HeLa cell growth was positively correlated with polyphenol content (Yuan and Walsh 2006). However, no correlation between growth inhibition and total phenolic content was found when extracts from three red seaweeds were tested against cancer cells, although there was a correlation with anti-oxidant effect (Zubia et al. 2009a, b). The growth of two tumour cell lines was inhibited and apoptosis increased by phlorotannin-rich extracts from Saccharina (Laminaria) japonica; anti-oxidant activity against several free radicals was also found (Yang et al. 2010a). Polyphenol-rich extracts were also found to inhibit the growth of colon cancer cells although there was no correlation with the phenolic content (Nwosu et al. 2011). Currently the evidence to support a correlation between the phenolic content of crude extracts and anti-cancer effects is limited, and claims should be made with care.

Induction of apoptosis by polyphenols

Although the cytotoxic effect of polyphenols is far from clear, some studies have shown that polyphenol-rich extracts can induce apoptosis in breast (MCF-7 and MDA-MB-231), colon (CT-26) and lung (A549) cancer cells. For example, a crude polyphenol extract induced apoptosis in colon cancer cells but had little effect on the viability of normal cells (Athukorala et al. 2006). In a study of three pure phlorotannin extracts, it was found that dioxinodehydroeckol showed the best dose-dependent cytotoxic effect against MCF7 and MDA-MB231 breast cancer cell lines. In MCF-7 cells, a number of pro-apoptotic effects were found including increases in PARP cleavage, p53, Bax and the activity of caspases 3 and 9. There was also a down-regulation of Bcl-2 nuclear factor kappa B (NF-κB) (Kong et al. 2009). Recently, a study of a crude polyphenol extract from the red seaweed Eucheuma cottonii (now Kappaphycus alverezii) has shown no effect against normal Vero cells. However, a dose-dependent decrease in proliferation and increase in apoptosis was found against MCF7 and MD-MBA-132 cells. In LA7 tumours grown subcutaneously in rats, the polyphenol-rich fraction also confirmed an anti-tumour effect; this included induction of apoptosis, down-regulation of endogenous oestrogen production and improvement of the anti-oxidative status of the rats (Namvar et al. 2012). In a review of the mechanisms underlying the role of polyphenols to TRAIL-induced apoptosis, it was claimed that polyphenols can restore tumour cell sensitivity to TRAIL-induced cell death with no apparent toxicity towards normal cells. There is evidence that both extrinsic and intrinsic pathways of apoptosis can be modulated by polyphenols, with activity depending on cell type, polyphenolic compound and experimental conditions (Jacquemin et al. 2010).

Anti-metastatic effect of extracts

There are few studies on the anti-metastatic effects of seaweed polyphenols on cancer cells. In one study of the polyphenolic compound 6,6′-bieckol extracted from E. cava, no significant inhibition of growth was found in HT1080 human fibrosarcoma cells. However, there was a change in cell morphology and reduced invasiveness that was accompanied by a reduction in matrix metalloproteinase-2 (MMP), MMP-9 and NF-κB (Zhang et al. 2010). MMP production is often deregulated in malignant tumours and is thought to influence tumour invasion and metastasis. Controlling its expression is therefore of interest since the results suggest that the compound may act to inhibit cell metastasis and migration, processes which are a main cause of morbidity and mortality in cancer patients.

Bromophenols

Work on bromophenols has been carried out on purified extracts as a rule, rather than on ‘phenol-rich’ crude extracts. Their bioactivity has been reviewed by Liu et al. (2011). An in vivo study of a crude extract from Leathesia nana, which was also purified to yield bromophenols, reduced S-180 tumour growth in Kumming mice (Shi et al. 2009). These results may provide a basis for further in vivo work with purified bromophenol compounds. Some mechanistic work has been recently published regarding a bis (2,3-dibromo-4,5-dihydroxybenzyl) ether, showing a possible pathway of action and binding site (Liu et al. 2012b).

Conclusions

The dual character of polyphenols as cytotoxic and anti-oxidant may explain why some researchers find that seaweed ingestion is cytoprotective since this could act to protect against DNA damage by free radicals. Attempting to elucidate the primary activity of polyphenols is therefore very difficult as the in vitro models are of limited value in elucidating the relevance of small effects and the in vivo models are difficult to study since the cancer protective effect will be small. The evidence suggests that depending on the extract, the dose and the model, polyphenols can be shown to exhibit both effects. It is unlikely that clear evidence on the anti-cancer properties of polyphenols will be available until the effects of purified extracts are investigated and even then the experimental model may be crucial. Human dietary trials could provide more evidence, but this approach has its own limitations (discussed in more detail below).

Carotenoids

Structure and occurrence

Carotenoids are natural pigment isoprenoid compounds found in both plants and animals. They are biosynthesised by tail to tail linkage of two C20 molecules to give a C40 molecule from which all carotenoid structures are derived (Britton 1995). In general, carotenoids are relatively easy to extract using a solvent and then saponification to remove impurities; they may then be purified by chromatography and are therefore often studied as pure or nearly pure extracts (Rodriguez 2001). However, isomerisation is common in carotenoids due to the presence of conjugated double bonds. In theory, every double bond in the chain may exist in the cis or trans form, giving rise to a huge number of possible configurations which provides an extra level of complexity even when the extracts are pure (Britton 1995). For example, the mixture of 13-cis and 13′-cis isomers of fucoxanthin extracted from U. pinnatifida had the greatest anti-proliferative effect compared to the other isomers tested; this was attributed to an increase in apoptosis induced in both HL-60 cells and Caco-2 cells (Nakazawa et al. 2009). The carotenoids astaxanthin and peridinin are found in microalgae and dinoflagellates and are not discussed here (for more information, see Palozza et al. 2009 and Sugawara et al 2007.)

Bioactivity

Various carotenoids have been shown to have both anti-cancer (Nishino et al. 1999) and/or anti-oxidant activity, whilst in certain conditions they may also show pro-oxidant effects (Young and Lowe 2001). The evidence for the bioactivity of carotenoids is primarily based on in vitro studies. However, it is likely that a number of factors reduce their effectiveness in vivo, making it difficult to extrapolate from in vitro studies (Young and Lowe 2001). Nevertheless, carotenoids including lutein, zeaxanthin, lycopene and β-cryptoxanthin have been shown to reduce carcinogenesis in vivo, and fucoxanthin has been shown to cause a marked reduction in tumour formation in mice (Nishino et al. 1999; Okuzumi et al. 1993).

Fucoxanthin is the most widely studied macroalgal carotenoid, and although it is found in many brown algae species, the majority of studies have investigated fucoxanthin extracted from one species U. pinnatifida (Table 7). Siphonaxanthin is found in siphonaceous green algae; to date, only two studies have reported on the bioactivity of this compound (Ganesan et al. 2010, 2011). Both fucoxanthin and siphonaxanthin have been found to induce cell cycle arrest and/or cell death via apoptosis in a variety of tumour cell lines derived from liver, prostate, colon, white cells and cervix; further characterisation has identified a range of pro- and anti-apoptotic mechanisms to be involved (summarised in Table 7). For example, fucoxanthin was found to cause DNA fragmentation and apoptosis in the prostate cancer cell lines, PC-3, DU145 and LNCaP (Kotake-Nara et al. 2001). In colorectal cancer cells, fucoxanthin induced a significant increase in apoptosis coupled with a decrease in Bcl-2 expression, and the viability of Caco-2 cells was synergistically reduced in combination with troglitazone, an inhibitor of the peroxisome proliferator-activated receptor γ (Hosokawa et al. 2004). In HL-60 leukaemic cells, fucoxanthin-induced apoptosis was linked to activation of caspases 3 and 9 with no effect on Bcl-xl, Bcl-2 or Bax (Kotake-Nara et al 2005a). However, in prostate cancer cells, fucoxanthin caused DNA fragmentation, caspase-3 activation and reduction in Bcl-2 and Bax (Kotake-Nara et al 2005b). In HepG2 and DU145 cells, fucoxanthin caused G1 cell cycle arrest which was later confirmed in LNCaP cells (Satomi and Nishino 2007; Satomi 2012).

It has been shown that fucoxanthin can be hydrolysed to fucoxanthinol during absorption both in colorectal adenocarcinoma cells (Caco-2) and in vivo in male ICR mice (Sugawara et al. 2002). A few studies have compared the effect of both compounds (Table 7). For example, treatment with either compound caused apoptosis and caspases 3, 8 and 9 activation in human T cell lymphotropic virus infected leukaemic cells (HTLV-1); however, neither affected uninfected cells (Ishikawa et al. 2008). Both compounds reduced proliferation in several tumour cell lines without blocking the toxicity of cisplatin (Mise and Yasumoto 2011), and both were also shown to induce apoptosis and cell cycle arrest (Yamamoto et al. 2011; Tafuku et al. 2012). When HL-60 cells were treated with either siphonaxanthin or fucoxanthin, apoptosis was increased accompanied by changes in GADD45α, DR5 and Bcl-2; the former caused more chromatin condensation and caspase activation (Ganesan et al. 2011). Only two studies have reported on the effect of macro-algal carotenoids on angiogenesis. Fucoxanthin and fucoxanthinol were found to suppress proliferation of HUVECs and decrease microvessel growth in ex vivo rat aortic rings (Sugawara et al. 2006). A similar study confirmed this result for fucoxanthin, and it also showed siphonaxanthin to have a comparable effect (Ganesan et al. 2010).

Conclusions

Clearly macroalgal carotenoids have potential as anti-cancer agents eliciting their effect primarily through a variety of apoptotic pathways and possibly in vivo through additional inhibition of tumour angiogenesis. One recent in silico study has suggested that fucoxanthin may cause cytotoxicity through a tubulin binding action (Januar et al. 2012).

Small molecules and lipids

Terpenes and derivatives

Terpenes are a large group of organic chemicals often found in plants that are composed of isoprene units arranged as chains or rings. In vitro studies have shown that terpenoid compounds often have good cytotoxicity towards tumour cell lines (Fuller et al. 1992; Ji et al. 2008), but normal cells are often also affected (Pereira et al. 2011; Campos et al. 2012) (see Table 8 for more examples). This shows the importance of testing the toxicity of compounds on normal as well as tumour cells. There have been few in vivo studies of terpenes and their derivatives; however, studies have been carried out on Caulerpa taxifola. One of the active compounds extracted from this seaweed, 10,11 epoxycaulerpenyne, was found to be toxic both to normal hamster kidney cells (BHK 21/C13) and Swiss mice (Lemée et al. 1993). An in vivo study of the sesquiterpenoid, elatol, showed that it could reduce growth of melanoma cells (B16F10) inoculated in C57BL6 mice (Campos et al. 2012).

Lactones and derivatives

Macrolides are a group of natural compounds characterised by the presence of a macrocyclic lactone ring. Details on their identification, isolation and cytotoxicity have previously been published (Kobayashi and Tsuda 2004; Tsuda et al. 2005). To the authors’ knowledge, no in vivo studies or assays on normal cells have been reported. However, the action of several macrolides on tumour cells in vitro has been reported, with many compounds showing promising IC50 values (see Table 8).

Steroids

Steroids are a class of organic compound characterised by a four-carbon ring structure. Sterols and stanols are subdivisions of steroids. No in vivo studies have been carried out in this sub-group of chemicals, but cytotoxicity against many tumour cell lines and some normal cell lines has been reported (see Table 8).

Alkaloids

Alkaloids are organic compounds which contain a basic nitrogen. Caulerpin has been isolated from several species of green and red algae (see review on alkaloids from marine algae, Güven et al. 2010). Lophocladines and caulerpin have shown cytotoxicity against a variety of tumour cell lines; however, caulerpin has also been shown to affect the growth of normal cells (Rocha et al. 2007; Liu et al. 2009a, b). When the crude extract, which was mostly composed of caulerpin, was compared to purified caulerpin from Caulerpa racemosa, only the crude extract showed cytotoxicity to melanoma cells; the authors suggested that this might be due to a synergistic effect (Rocha et al. 2007).

Quinones

Quinones have been mostly found in brown algae, although a few have been isolated from green algae. Their cytotoxicity has been recently reviewed (Sunassee and Davies-Coleman 2012). Quinone compounds have been found to be cytotoxic to tumour cells, with some cytotoxicity seen in normal cells where tested (Perry et al. 1991; Iwashima et al. 2005). A mechanistic study has shown that sargachromanol E, a compound with a quinone moiety, caused apoptosis in HL-60 cells.

Lipids

Some uncharacterised lipids, as well as fully characterised molecules, have been extracted from macroalgae. Cytotoxicity was found against a variety of tumour cell lines, although no comparisons to normal cells have been reported (see Table 8). One in vivo study of uncharacterised lipid fractions showed good anti-tumour activity against Meth A fibrosarcoma implanted in BALB/c mice (Noda et al. 1989).

Conclusions

Molecules in this category are usually very well characterised, but in most studies the anti-cancer cytotoxicity is mainly studied as part of a screen when testing for other bioactivities, e.g. anti-microbial, anti-inflammatory etc. Molecules which have provided promising results require further experimentation to determine the mechanism of their cytotoxicity and also if they have any effect on normal cells. In vivo studies should then be carried out to identify differential toxicity, an essential step in the development of anti-cancer drugs for humans.

Protein and peptides

A recent review has emphasized the potential of peptides, proteins and amino acids from macroalgae as a rich source of bioactive molecules (Harnedy and FitzGerald 2011). However, there are currently only a limited number of studies on proteinaceous extracts as a potential source of anti-cancer agents (Table 9). In general, these extracts show good activity against tumour cells with little or no activity against normal cells (Sugahara et al. 2001; Go et al. 2009; Kim et al. 2012b). One early study showed toxicity against the ‘normal’ CV1 monkey fibroblast cells of kahalalide F, a peptide isolated from the seaweed Bryopsis pennata and in greater quantities from the mollusk Elysia rufescens which feeds on it. However, kahalalide F also showed efficacy against a number of tumour cell lines (Hamann and Scheuer 1993), and this potency resulted in it entering clinical trial. The drug was well tolerated and showed a good safety profile (Pardo et al. 2008) underlining the limitations of extrapolating from normal cell line toxicity data to animal models and human trials. The drug was then tested in a phase II trial of cutaneous malignant melanoma; it was again well tolerated but showed no significant anti-tumour activity resulting in early closure of the trial (Martín-Algarra et al. 2009). A new phase I trial to determine the safety of prolonged infusions in order to achieve longer exposure time was carried out. A dose with an acceptable safety profile was found, and this will allow a phase II trial to be carried out in the future (Salazar et al. 2013). Clearly peptide/protein fractions of macroalgae are under-researched despite their potential exemplified by kahalalide F which is the only macroalgal product to have entered clinical trials.

Crude extracts

In vitro studies

There have been many studies of the anti-cancer properties of seaweeds using unpurified fractions from a variety of solvent/water combinations; these have shown a range of anti-tumour effects (Table 10). For example, in a study of several seaweed species, dichloromethane/methanol extracts were found to be more cytotoxic than water extracts (Moo-Puc et al. 2009). Methanol, rather than water extracts, had the most potent telomerase inhibiting activity (Kanegawa et al. 2000). Methanol extracts of the brown seaweeds Sargassum swartzii, Cystoseira myrica and Colpomenia sinuosa were partitioned to obtain different fractions in hexane, chloroform, ethylacetate and methanol/water; the hexane fractions were found to be the most effective (Khanavi et al. 2010). Hexane extracts from Laurencia sp. were also found to be most cytotoxic to uterine and cervical cancer cell lines (chloroform and methanol extracts were also tested) (Stein et al. 2011). In a recent study, the ethyl acetate fraction of Cytoseira compressa was shown to be the most cytotoxic, followed by the chloroform and methanol fractions (Mhadhebi et al. 2012).

Enzyme targets in cancer cells have also been investigated. A comprehensive screen of 304 seaweed samples, collected from diverse sites around the Japanese archipelago, investigated their ability to inhibit telomerase in MOLT-4 cells using methanol and water-based extracts. Twelve samples showed inhibitory activity with a methanol extract from the green alga Caulerpa sertularioides being the most effective (Kanegawa et al. 2000). Telomerase is recognised as an attractive target in anti-cancer therapy as the enzyme is frequently up-regulated in cancer cells, a characteristic associated with cells that can proliferate indefinitely (Shay and Wright 2006). Protein kinase A is another enzyme which has potential as a target for inhibiting cancer cells. In a pilot screen of marine macroalgae collected in Australia, uncharacterised ethanol extracts of Porphyra sp., Ecklonia radiata and Sargassum vestitum were shown to be potent inhibitors of protein kinase A (Winberg et al. 2011). Other in vitro studies are summarised in Table 10.

In vivo studies

Early in vivo studies using crude preparations from a variety of seaweed species, including Laminaria species, showed anti-tumour efficacy as shown by decreases in tumour size and incidence (Yamamoto and Maruyama 1985; Yamamoto et al. 1986). A study on 7,12-dimethylbenz(α)anthracene (DMBA)-induced skin tumours in ICR mice showed that a dichloromethane extract from the brown seaweed U. pinnatifida reduced the number of tumours compared to the control (Ohigashi et al. 1992). When the toxicity of crude water and methanol extracts from Caulerpa taxifolia was studied on Swiss mice, seasonality was found to have an effect. Water extracts were found to be more toxic when isolated in the winter and spring, and methanol extracts were more toxic in summer (Lemée et al. 1993). The development of chemically induced skin tumours was reduced in ICR mice by a methanol/acetone extract from the green seaweed Enteromorpha prolifera (Higashi-Okai et al. 1999). Inhibition of metastasis was demonstrated in vivo with a water extract from the red algae Marginisporum crassissimum (Hiroishi et al. 2001). Ethanol extracts from L. nana were able to reduce growth of S-180 tumours in mice (Shi et al. 2009), and ethanol extracts from Acanthophora spicifera were able to decrease the volume and weight of Erlich ascites carcinoma in mice (Lavakumar et al. 2012). A study on gastric cancer induced in rats showed anti-oxidant activity and immune stimulation on treatment with a water extract from S. pallidum (Zhang et al. 2012).

Effect on normal cells

In general, where normal cells have been studied in vitro, crude extracts have been shown to be less toxic to normal cells than to tumour cells (Funahashi et al. 2001; Lee et al. 2004; Moo-Puc et al. 2011b; Rocha et al. 2007; Lin et al. 2012). An extract from Ulva lactuca was found to stimulate growth of splenocytes, whilst inhibiting tumour cell growth, indicating the possibility of an anti-cancer agent with potential to stimulate the immune system (Lee et al. 2004).

Conclusion

Clearly many seaweeds show potential as a source of novel anti-cancer agents, and in some cases, specific targets have been identified. However, studies with crude extracts need refining if the active compound(s) are to be identified, and in many cases, further purification can be problematic.

Raw/processed seaweed

Animal nutrition studies

The majority of studies that involve feeding seaweed to animals have investigated the putative chemoprotective effect of raw seaweed when the animals are exposed to carcinogenic agents. In many studies, it has not been noted whether fresh or dried seaweed has been used. A number of these studies are summarised in Table 11; studies that involve investigation of the therapeutic potential of seaweed and seaweed extracts have been discussed above (also see Tables 4, 5, 6, 7, 8, 9 and 10).

Table 6 Polyphenols and brominated phenols from seaweeds with anti-cancer potential
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Table 7 Carotenoids from seaweeds with anti-cancer potential
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Table 8 Small molecules from seaweeds with anti-cancer potential
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Table 9 Proteins and peptides from seaweeds with anti-cancer potential
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Table 10 Crude extracts from seaweeds with anti-cancer potential
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U. pinnatifida (wakame) suppressed the growth of chemically induced mammary tumours in mice. It was fed to the mice at levels of 1 and 5 % in normal feed. The authors suggest a connection between iodine content and reduction in proliferation of cancer cells. Iodine had previously been found to reduce the proliferation of breast cancer cells in vitro and in vivo (Funahashi et al. 1999). In a letter to the editor, Tokudome et al. (2001) emphasise that caution should be exercised in hypothesising which component has the anti-cancer effect. In addition, they point out that excessive consumption of seaweed can lead to an excess of iodine in the diet, which can cause diseases such as thyrotoxicosis in humans.

Human nutrition studies

Most human studies have involved epidemiological analysis of cancer rates in populations eating seaweed as a part of their standard diet. In most of these studies, there is no further clarification as to the seaweed species eaten, or indeed the method of processing which can vary considerably and may also include further cooking by the consumer; all of these will affect the bioactive components. For example, an investigation of Himanthalia elongata (B) showed how drying affected its anti-oxidant profile (Gupta et al. 2011).

Along with preparation and cooking, it is well recognised that there are many factors that can influence dietary studies which makes interpretation difficult. There is also an important dichotomy of effect that needs to be considered when investigating the effects of seaweed ingestion. Firstly, they can potentially act in a protective manner to stop initiation or promotion of tumours, and in addition, the presence of natural cytotoxins could kill small foci of latent tumours either directly or by enhancing immune surveillance mechanisms. This is highlighted in a review on the effect of Laminaria on breast cancer in which the author discusses a number of mechanisms through which Laminaria can affect breast cancer rates. For example, the seaweed is a good source of fibre which increases faecal bulk and decreases bowel transit time, and it contains anti-bacterial activity that will influence faecal microflora. It also affects sterol metabolism, and it stimulates the host-mediated immune response all of these have the potential to reduce cancer rates (Teas et al. 1982).

One of the early reports on the influence of diets high in seaweed was a study of a group (>8,000) of native-born and first-generation Japanese men living in Hawaii. In a later sub-analysis of the wives of these men, it was found that 86 had a confirmed diagnosis of breast cancer and that there was a significantly lower incidence of breast cancer in the wives who ate a more Japanese style diet which was high in seaweed. However, this interpretation is based on a reasonable, but unproven, assumption that the women ate a similar diet to the men who were the study respondents (Nomura et al. 1978). In another population of ethnic Japanese living in Hawaii, the incidence of prostate cancer was found to be increased in those eating five or more helpings of seaweed per week suggesting that moderation in seaweed ingestion might be a better option (Severson et al. 1989). A study has been reported from the Saitama Prefecture in Japan where lifestyle and dietary interviews were carried out with 181 patients recently diagnosed with colorectal cancer and 653 controls obtained from the local population. A few significant correlations were found including an independent inverse association between seaweed consumption and both colon and rectal cancer (Hoshiyama et al. 1993).

The JACC study investigated a very large cohort of Japanese men (n = 42,940) and women (n = 55,308) aged 40 to 79 years during 1990–1997. Seaweed consumption, amongst many other foods, was assessed using a self-administered food frequency questionnaire. During this period, 446 males and 126 females died from lung cancer (information obtained from death certificates). Several associations were found including an inverse correlation between lung cancer and seaweed consumption in men, although this was not significant for women possibly due to the small reported incidence. This underlines the difficulty in obtaining clear information from dietary studies. Even in this study over 7 years and nearly 100,000 participants, only a few trends were found and they varied by gender (Ozasa et al. 2001). As discussed above, excessive seaweed consumption can increase iodine levels, and this can cause diseases such as thyrotoxicosis which is especially true for individuals who are unaccustomed to a high iodine diet (Tokudome et al. 2001).

Recently, a significant inverse correlation was found between breast cancer rates and Porphyra (gim or nori) consumption in Korean women, whereas no significant correlation was observed with U. pinnatifida (miyeok or wakame) intake. However, the authors recommend further work and point out the limitations of the study including low consumption in the groups and low variation between cases (Yang et al. 2010b). In a large, recently reported prospective study (n = 52,679) supported by the Japan Public Health Centre, a positive association was found between seaweed consumption and thyroid cancer risk (especially papillary carcinoma) in postmenopausal but not premenopausal women. Hazard ratio for almost daily consumption compared with 2 days/week or less was 1.71; 95 % CI 1.01–2.90; trend P = 0.04.

The difficulty of interpretation of dietary studies has been further emphasised by an interventional study of breast cancer risks in American women. The study was designed to investigate the potential effect of seaweed on soy-associated increases in IGF-1 since increased IGF-1 is associated with an increased risk of post-menopausal breast cancer. The study confirmed that soy significantly increases serum IGF-1 levels; however, combination with seaweed reduced this increase by about 40 %. This suggests that concurrent seaweed and soy consumption may be important in modifying the potential deleterious effect of soy-induced increases in serum IGF-1 (Teas et al. 2011). A further preliminary dietary study showed that seaweed supplementation (U. pinnatifida capsules) reduced the concentration of urinary human urokinase-type plasminogen activator receptor (uPAR) in postmenopausal American women, an effect that was reversible after seaweed supplementation ended. It is suggested that this may explain the reduced prevalence of breast cancer in Japanese women, where seaweed is frequently consumed, as a raised uPAR level is known to be associated with a higher incidence of breast cancer and an unfavourable prognosis (Teas et al. 2013). This is an important finding as to the potential of seaweed in protecting against BC; however, this study was not designed to identify any potential of the Undaria capsules as a source of novel CCT.

Conclusions

Dietary studies provide a unique challenge when trying to identify the efficacy of individual components. In animals, these studies have primarily been confined to situations where seaweed has been tested as a means to reduce tumour development in response to toxic levels of tumour initiators and/or promoters. In humans, tumours develop at a very much reduced rate and over a longer timespan. Human dietary studies do show, on average, trends in favour of seaweed ingestion in the protection from cancer development but the effect is small. Caution must also be exercised since too much seaweed in the diet can increase iodine ingestion to toxic levels. Also there is some evidence that prostate cancer rates may be increased in men eating high levels (≥5 portions/week) of seaweed; thus, moderation may be the best policy. In view of the considerable limitations on the type of studies possible in animal models and particularly humans, it may always be difficult to draw conclusions on the potential use of seaweed as a source of novel anti-cancer agents. The only exception to this would be a fully powered phase 3 clinical trial of a potentially effective cancer drug that has progressed over all of the hurdles to this final stage before licensing.

Overall conclusions

Over the last 40 years, there has been an increasing interest in the identification of novel drugs isolated from natural compounds including macroalgae. The papers identified in this review (Tables 4, 5, 6, 7, 8, 9, 10, 11 and 12) show that many different seaweeds have been investigated, primarily sourced from the seas off the coasts of Asia but including diverse sites from around the world. To fully identify the potential of macroalgae, there is a need to expand these studies to seaweeds collected in the seas off other continents since it is known that there is great spatial diversity in both species of seaweed and the bioactive molecules they contain.

Table 11 Raw seaweeds with anti-cancer potential
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Table 12 The effect of seaweed ingestion in humans, using epidemiological and interventional methodology
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Many of the studies discussed have identified good to excellent potential of the macroalgae as sources for anti-cancer drugs; this has been proven in a range of different models both in vitro and in vivo. However, very few preparations, from any marine sources, have made it to clinical trial, and only one seaweed-derived drug, kahalalide F, has been tested (currently in clinical trials, see “Protein and peptides”).

One of the problems of natural product research is the purification and identification of the active compound(s). Most studies use crude extracts or partially purified fractions. This approach is useful when screening for bioactivity, and any activity found is frequently attributed to the predominant component of the extracts. However, this cannot be confirmed since there may be other less abundant, but more potent compounds, in the extract. Clearly, when potent effects are found, there is a need for further work to fully purify and characterise the extracts so that activity can be attributed to a specific class of compounds or a molecule and the mechanism of action identified. However, this is not a simple matter and takes considerable levels of investment which perhaps explains why so many apparently effective crude fractions have failed to be developed.

An additional complication is the potential for synergistic and/or antagonistic effects of crude extracts to be lost on purification, and this may not become apparent until complex purification has taken place. Also when testing for compounds that have cytotoxic effects on cancer cells, it is essential that there is tumour specificity; this requires the concurrent study of normal cells and evaluation of side effects in animal models and humans. In addition, in recent years, the regulatory authorities require more specific information as to how a new drug works, and this involves carrying out mechanistic studies to identify the specific targets in cancer cells which are less affected in normal cells. Some studies have attempted to do this, and a variety of pathways have been implicated most frequently those controlling apoptosis.

As discussed above, human dietary studies suggest that seaweed in the diet has a protective effect against tumour development, although there are some suggestions that too much can have deleterious effects in some situations. The human studies primarily measure the protective effect of dietary seaweed on humans, and they do not really inform as to whether they contain potential cytotoxic compounds. However, this may be one of the mechanisms through which they may work, by reducing the viability of micro-tumours or enhancing the immune system’s ability to eradicate small, invisible clusters of tumour cells before they become clinically demonstrable; currently, it is not possible to test this.

This review has compiled considerable evidence that marine-derived macroalgae are an excellent source of compounds with the potential to be developed into drugs for treating cancer. However, considerable investment is required if this valuable resource is to be utilised to its full potential.