**3. Fucoidans and Metastasis**

In cancer, many cells develop the ability to invade adjacent tissue components of its primary organ and spread to other organs [25]. This process is called metastasis and involves several steps including altered cellular adhesions, cell motility, resistance to extracellular death signals, and disruption of the basement membrane and ECM [26]. Metastasis is responsible for more than 90% of cancer deaths [27] due to its systemic nature and higher drug resistance. Therefore, new molecular or clinical strategies are needed to counteract this aggressive feature [28]. In general, the metastasis process can be divided into 4 steps: (1) Certain tumor cells obtain characteristics of epithelial–mesenchymal transition (EMT), dissociating and detaching from the primary tumor to escapes from this area. (2) The dissociated tumor cells infiltrate into the surrounding stroma and invade and migrate through the basement membrane supporting the endothelium of local blood and/or lymphatic vessels. (3) The dissociated tumor cells cross the ECM resulting in intravasation. This involves dissemination of tumor cells to distant organs through blood or lymph vessels. These tumor cells can then forma new tumor in other organs or tissues (secondary tumor) through mesenchymal to epithelial transition (MET), which is another mechanism that enables metastatic colonization (neoplasm) and that is the contrary to EMT (e.g., re-expression of E-cadherin). (4) The final dormancy step is characterized by invading tumor cells that can remain silent for many years in the distant organ [29]. Both step 1 (EMT) and 2 (infiltration and invasion into stroma) are characterized by morphological changes from the epithelial cell monolayer with an apical-basal polarity, to dispersed, spindle-shaped mesenchymal cells with migratory protrusions [30]. In particular, EMT involves changes in the expression of cell–cell junction proteins, cytokeratin intermediate filaments, increase vimentin filaments and fibronectin [31]. In this case, sulphated fucoidans have been shown to maintain the endothelium adhesion by binding to endothelial cell receptors, especially when the polysaccharides that normally bind to these receptors decrease, confirming that fucoidans have antimetastatic effects and can prevent EMT [32]. A recent study demonstrated this using fucoidan from *F. vesiculosus*, which was able to inhibit the EMT and, therefore, an important step in the metastasis development [33]. In addition, fucoidan has been shown to decrease the activity or expression of transforming growth factor receptors (TGFRs) in vitro and in vivo. This blocks the EMT process and its morphological changes by upregulating epithelial markers, downregulating mesenchymal markers and decreasing the expression of transcriptional repressors such as SNAIL, SLUG, and TWIST, which subsequently induce migration and invasion inhibition [34]. Moreover, fucoidans are also able to reduce TGFR downstream signaling events, including SMAD2/3 and non-SMAD pathways: AKT, ERK1/2, and Focal Adhesion Kinase (FAK) phosphorylation. Fucoidans decrease TGFR proteins by ubiquitination proteasome pathway (UPP)-mediated degradation of TGFRs and by the promotion of SMURF2 and SMAD7 that conjugate to TGFRs, resulting in TGFR degradation [35].

Post-transcriptional mechanisms have also been implicated in the control of EMT and their relationship to TGF-β signaling through microRNAs (miRs). In this context, fucoidan of *S. hemiphyllum*, increases the miR-29 family expression that suppresses *DNMT3B* expression, which results in the

upregulation of the tumor suppressor gene *MTSS1*. This fucoidan also downregulates TGF-β signaling, increases E-cadherin expression, decreases N-cadherin, *ADAM12*, and *PTEN* expression, and finally prevents ECM degradation by overexpressing *TIMP-1* and reducing the expression of matrix metalloproteinase enzymes MMP2 and MMP9, secreted by cancer cells to degrade ECM and induce cell migration [36,37]. Furthermore, an oligo-fucoidan extracted from *S. hemiphyllum* has been shown to inhibit the signaling of chemokine CCL2, which has a chemoattractant activity for monocytes, T cells, mast cells and basophils, and promotes invasion and metastasis via JAK-STAT and MAPK signaling pathways. Therefore, this CCL2 inhibition induces an inflammatory response, anti-tumor immunity and tissue conservation to avoid metastasis and angiogenesis [18]. Another example is the fucoidan of *S. fusiforme* which has an antimetastatic effect on liver cancer cells by inactivating the integrin αVβ3 and prevent the invadopodia formation [38].

Another characteristic of metastasis is the involvement of cell migration and invasion properties through ECM [30]. As fucoidans have structural similarities with heparin, these polysaccharides not only have anticoagulant features but also are able to decrease the expression and activity of matrix metalloproteinases, resulting in an incapability of tumor cells to cross the capillary wall [39]. For example, fucoidan derived from *Undaria pinnatifida sporophylls* inhibits in vitro cell growth, migration, invasion, and adhesion capabilities probably by downregulating the VEGFC/VEGFR3 axis, inactivating the NF-kB pathway and increasing the protein levels of TIMPs [40]. Other fucoidans decrease the expression levels of MMP2 in a dose dependent manner and downregulate the PI3K/Akt/mTOR signaling pathway [41].

Fucoidan of *Laminaria japonica* reduce the migratory and invasive features of triple-negative breast cancer (TNBC) cell models by suppressing the activation of MAPK and PI3K pathways and subsequently inhibiting AP-1 and NF-κB signaling. Additionally, this fucoidan was shown to inhibit micrometastasis in an in vivo transgenic zebrafish model [42].

Hypoxia in tumoral microenvironment is another phenomenon that can lead to metastasis. Fucoidan derived from *U. pinnatifida sporophylls* inhibit hypoxia in cancer cells through nuclear translocation, activity of HIF-1α and reduction in the levels of phosphorylated-PI3K (p-PI3K), p-Akt, p-mTOR, p-ERK, NF-κB, MMP-2, and MMP-9, but increased TIMP-1 levels. In addition, this fucoidan can decrease the levels of VEGF-C and HGF [43]. The most complete studies about inhibition of metastasis and drug resistance by fucoidans are shown in Table 1 and the main signaling pathways involved in these processes are shown in Figure 2.

Given the biological activities and implications of fucoidans in cancer, particularly in metastasis, the sulphated polysaccharides are candidates to generate functional foods and drugs as well as for their applications in prevention, synergism with chemotherapy, and nanotechnology. For instance, one nanotechnology application is the utilization of polysaccharides by eco-friendly synthesis of fucoidan-stabilized gold nanoparticles for charge interaction [44]. This demonstrates the potential of fucoidan to be used as a therapeutic agen<sup>t</sup> and as technological material.


**Table 1.** Sources, characteristics and effects of fucoidans on the metastatic and drug-resistant phenotype of cancer models.


**Table 1.** *Cont.*


**Table 1.** *Cont.*


**Table 1.** *Cont.*

growth factor. EMT. Epithelial-mesenchymal transition. ER: Estrogen receptor. FAK: Focal adhesion kinase. FE: Fucoidan extract. GSH: Glutathione. HCC: Hepatocellular carcinoma. HGF: hepatocyte growth factor. CRC: colorectal cancer. NSCLC: Non-small-cells human bronchopulmonary carcinoma. IC-ROS: Intra cellular reactive oxygen species. LMWF: Low molecular weight fucoidan. MMP: Matrix metalloproteinase. NDRG: N-myc downstream-regulated gene. PTEN: phosphatase and tensin homolog. PVR: Proliferative vitreoretinopathy. ROS: Oxygen reactive species. RPE: Retinal pigment epithelial. TGFR: Transforming growth factor-b receptor. TIMP: Tissue inhibitor of metalloproteinase. VEGF: Vascular endothelial growth factor. VMP: vacuole membrane protein.

**Figure 2.** Summary of the main signaling pathways involved in the fucoidan function during the processes of metastasis and drug resistance.

#### **4. Fucoidans and Drug Resistance in Cancer**

There are many types of cancer treatments, including surgery, radiation, chemotherapy, hormone therapy and, more recently, target therapy (e.g., chemokine receptors), stem cells transplantation, and immunotherapy [67]. One of the major complications in cancer treatment is the appearance of chemotherapy resistance, which is defined as the development of innate and/or acquired ability by cancer cells to evade the effects of chemotherapeutics [68]. Some cancer cells are intrinsically resistant to chemotherapy and others are able to develop a resistance phenotype, either by their own characteristics as tumor cells or by external conditions such as the tumor microenvironment [69]. For instance, repeated chemotherapeutic stimulation can induce pro-survival biological changes in tumor cells, allowing them to evade cell death under drug pressure by using host or tumor-related factors [70]. Most chemotherapeutic agents in cancer therapy (e.g., platinum drugs, taxanes) induce cell stress on "sensitive cells" resulting in cell death mediated predominantly by the apoptosis pathway [71]. Despite the effectiveness of programmed cell death induced by drugs, because tumors are heterogeneous in nature, certain cancer cells can display a drug-resistant behavior. This constitutes the main obstacle for anticancer therapeutic success [72]. There are four major mechanisms that contribute to drug resistance in cancer cells: (1) Decreased uptake of water soluble drugs [73]; (2) changes in intracellular pathways that affect the potential of cytotoxic drugs to kill cells, including alterations in the cell cycle, DNA repair, apoptosis pathways, metabolism/elimination of drugs, or others [73–75]; (3) increased energy-dependent efflux of hydrophobic drugs mediated via overexpression of a family of energy-dependent transporters (known as ATP-binding cassette transporters) such as P-glycoprotein 1 (P-gp, ABCB1) or breast cancer resistance protein (ABCG2) amongs<sup>t</sup> others [73]; and (4) intracellular detoxifiers such as antioxidants (e.g., glutathione) [76,77]. Multiple signaling pathways have been implicated in resistance to chemotherapy, and innovative therapeutic strategies to overcome these are urgently needed [78].

Some fucoidans have been implicated in the decrease of the cancer drug resistant phenotype (Table 1). For example, fucoidans from *A. nodosum* showed an arrest in G1 phase of the cell cycle and a reduction in the chemoresistance to cisplatin of non-small-cell human bronchopulmonary carcinoma (NSCLC-N6) cells, a type of chemoresistant cell line [62]. The same study also showed an antitumor effect at sub-toxic doses of fucoidan in vivo in NSCLC-bearing nude mice [62]. Similarly, a sulphated

fucan-like polysaccharide with aminosugar obtained from *Turbinaria ornate* was shown to arrest cell cycle in G1 phase in NSCLC-N6 cells [63]. A fucoidan obtained from *F. veciculosus* was able to decrease the expression of cellular prion protein (PrPC) HT29 colon cancer cell lines. PrPC is a protein whose overexpression is involved in increasing cell survival and proliferation, and inhibition of stress-response proteins p38, JNK, and p53, which could induce drug resistance [54,79].

More recently, cytokines have been shown not only to directly influence cancer progression by inducing cancer cell proliferation, migration, metastasis, reprogramming of tumor microenvironment (TME), immune evasion and the formation of new blood vessel within tumors [80,81] but are also often associated with chemoresistance and overall poor prognosis [80,82–86]. In this context, certain oligo-fucoidan have been shown to produce pro-inflammatory cytokines and chemokines (e.g., IL-6 and CCL2/MCP-1 respectively) and decrease the side e ffects of chemotherapy [18]. Also, other fucoidans can downregulate some cytokines and chemokines (e.g., M2-type chemokine CCL22) to inhibit tumor cell migration and lymphocytes recruitment via NF-κB-dependent transcription, which may be a novel and promising mechanism for tumor immunotherapy [46].

Fucoidans can also function as adjuvant agents along with chemotherapy. For instance, it has been demonstrated that sulphated polysaccharides can increase the bioavailability of certain oral drugs, like doxorubicin [87]. Fucoidans from *U. pinnatifida* and *F. vesiculosus* have been studied in combination with tamoxifen and paclitaxel in orthotopic mouse models of breast cancer and ovarian cancer. The results showed that both fucoidans improved the e ffect of tamoxifen, but not paclitaxel, in breast cancer. In the ovarian cancer model, only fucoidan from *F. vesiculosus* was able to improve the activity of tamoxifen, but not paclitaxel [50]. Fucoidan from *F. vesiculosus* has been shown to increase cytotoxicity of cisplatin on lung cancer cell lines via upregulation of cleaved caspase-3 and poly (ADP ribose) polymerase (PARP) expression, which induces apoptosis in these cells [47]. In addition, this fucoidan can also act synergistically with gefitinib to induce apoptosis in lung cancer cells [48].

Fucoidan from *U. pinnatifida* has also been investigated in melanoma, which is an intrinsically aggressive and therapy-resistant cancer that can develop resistance to the ERBB inhibitor, lapatinib. While, lapatinib alone inhibited 60% of tumor growth, in combination with fucoidan it decreased 85% of tumor growth. In addition, the use of fucoidan can counteract the morbidity associated with prolonged lapatinib treatment. This ability to avoid side e ffects provides an additional advantage for the potential use of fucoidan extracts [59]. Another fucoidan extracted from *Cladosiphon navae-caledoniae* Kylin in combination with cisplatin, tamoxifen or paclitaxel can improve outcomes in breast cancer treatment. These co-treatments significantly inhibited cell growth in MDA-MB-231 and MCF-7 breast cancer cells. Furthermore, they enhanced apoptosis in these cells by downregulating anti-apoptotic proteins Bcl-xL and Mcl-1 and promoting higher intracellular ROS levels [58].

Fucoidans have particular chemical characteristics (backbone with fucose sugar and sulphate group) that confer them a negative surface and favor interaction with other chemical compounds or cellular molecules. This makes them an interesting material for the development of nanoparticles. Hwang et al. designed fucoidan-cisplatin nanoparticles with high cisplatin content and loading efficiency. These were used to treat macrophage cells (RAW264.7) to assess immune protection from the cytotoxicity of cisplatin [88]. Indeed, the cells treated with fucoidan-cisplatin conjugation were more protected in comparison to cells treated with cisplatin alone. Moreover, the fucoidan-cisplatin nanoparticles showed stronger cytotoxicity against colon cancer cell lines than those treated with cisplatin alone, which suggests that fucoidan-based nanoparticles with high drug encapsulation have a potential application in immunotherapy and chemotherapy [88]. Other nanoparticles with fucoidan-coated manganese dioxide were applied in pancreatic cancer cell models associated to hypoxia as a mechanism of resistance to radiation therapy [56]. The nanoparticles not only showed a significant decrease of HIF-1 expression under a hypoxic condition, but they were also able to reverse hypoxia-induced radioresistance. The latter was shown by a decrease of clonogenic survival and an increase of DNA damage and apoptosis in response to radiation therapy. In vivo studies showed that fucoidan-coated manganese dioxide nanoparticles along with radiotherapy also decrease tumor growth in comparison to radiation alone [56]. Therefore, fucoidan-coated manganese dioxide nanoparticles have clinical potential in the treatment of hypoxic, radioresistant pancreatic cancer [56] (Figure 2). Furthermore, a combinational synergistic e ffect between fucoidan (natural compound), doxorubicin (chemotherapeutic drug) and photothermal nanocarrier (Pt nanoparticle) has been observed as it was possible to reverse the drug resistance of breast cancer cells submitted to photothermal therapy [66]. In this case, the fucoidan was applied as a biocompatible surfactant and surface-coating biopolymer in the fucoidan-coated photothermal nanocarrier. As a result, the biological–chemo–thermo combination treatment showed a promising therapeutic e fficiency against multidrug resistant breast cancer cell MCF-7 ADR both in in vitro and in vivo breast cancer models [66]. Fucoidan from *F. vesiculosus* assembled within nanoparticles bearing doxorubicin improved significantly the chemotherapy response in breast cancer cell lines by enhancing their immunostimulatory activity [51].

The molecular mechanisms of drug resistance have been classified into pre-target (alterations that precede the binding to DNA), on-target (alterations that are directly related to drug-DNA interaction), post-target (mechanisms downstream of DNA damage with e ffect in cell death signaling pathways) and o ff-target (influencing on molecular processes that are not directly associated with drug-elicited signals) [78]. In this context, the potential mechanisms in which fucoidans can reverse the drug resistance are versatile. Fucoidans can inhibit chemokine/chemokine receptors interaction as a pre-target mechanism [18]. The increase of cell cytotoxicity and arrest of the cell cycle demonstrates their e ffect on on-target mechanisms [62].They can influence post-target mechanisms, for example through the downregulation of anti-apoptotic proteins Bcl-xL and Mcl-1.and finally, the promotion of higher intracellular ROS levels, is an example for their role in an o ff-target mechanisms [58].
