The Combined Metabolically Oriented Effect of Fucoidan from the Brown Alga Saccharina cichorioides and Its Carboxymethylated Derivative with 2-Deoxy-D-Glucose on Human Melanoma Cells

Melanoma is the most aggressive and treatment-resistant form of skin cancer. It is phenotypically characterized by aerobic glycolysis that provides higher proliferative rates and resistance to cell death. The glycolysis regulation in melanoma cells by means of effective metabolic modifiers represents a promising therapeutic opportunity. This work aimed to assess the metabolically oriented effect and mechanism of action of fucoidan from the brown alga Saccharina cichorioides (ScF) and its carboxymethylated derivative (ScFCM) in combination with 2-deoxy-D-glucose (2-DG) on the proliferation and colony formation of human melanoma cell lines SK-MEL-28, SK-MEL-5, and RPMI-7951. The metabolic profile of melanoma cells was determined by the glucose uptake and Lactate-GloTM assays. The effect of 2-DG, ScF, ScFCM, and their combination on the proliferation, colony formation, and activity of glycolytic enzymes was assessed by the MTS, soft agar, and Western blot methods, respectively. When applied separately, 2-DG (IC50 at 72 h = 8.7 mM), ScF (IC50 at 72 h > 800 µg/mL), and ScFCM (IC50 at 72 h = 573.9 μg/mL) inhibited the proliferation and colony formation of SK-MEL-28 cells to varying degrees. ScF or ScFCM enhanced the inhibiting effect of 2-DG at low, non-toxic concentrations via the downregulation of Glut 1, Hexokinase II, PKM2, LDHA, and pyruvate dehydrogenase activities. The obtained results emphasize the potential of the use of 2-DG in combination with algal fucoidan or its derivative as metabolic modifiers for inhibition of melanoma SK-MEL-28 cell proliferation.


Introduction
Melanoma is a highly aggressive form of skin cancer with an increasing prevalence worldwide [1]. In 2020, an estimated 325,000 new cases of melanoma were diagnosed all over the world, and 57,000 people died from the disease [2]. Even with recent advancements in melanoma therapies, namely, immunotherapies, such as ipilimumab, targeted therapies, such as vemurafenib, or combination therapies, such as polychemotherapy, polyimmunotherapy, and biochemotherapy, the management of advanced melanoma is very challenging [3][4][5][6]. The highly proliferative and invasive phenotypes of melanoma cells exhibit an increased demand for energy and building blocks and, consequently, reprogramming of cellular metabolism [7]. The malignant state of melanoma is associated with higher glycolytic activity and utilization of glucose and with lower mitochondrial respiration, even under normoxic conditions [8,9]. This metabolic profile is known as the Warburg effect and is a hallmark of many cancer types [10]. Therefore, interfering with the metabolism of melanoma cells may be a promising effective therapeutic strategy that may help to improve the existing standard therapies. It was previously found that S. cichorioides produces predominantly fucoidan with a backbone of mainly (1→3)-linked α-L-fucopyranose residues and a small amount of (1→4)linked fucopyranose and branches at position 2 in the form of single α-L-fucose residues. Sulfate groups were found at positions 2 and 4 [25]. An NMR spectroscopy confirmed that the ScF obtained in the present study had a similar structure (Supplementary Figure S1a).
Modification of polysaccharides is one of the promising ways of obtaining new compounds for medicine, chemistry, ecology, and industry. In the present study, the carboxymethylation of ScF was based on the reaction of the O-alkylation of the polysaccharide with acetic acid in NaOH and isopropanol.
We studied the structure of the obtained polysaccharide, ScFCM, via 13 C NMR spectroscopy (Supplementary Figure S1b). It confirmed that this derivative contained car-boxymethyl groups. Thus, the signals belonging to the carbonyl group at 178.8 ppm and to the methylene carbon at 71.1 ppm appeared in the spectrum after the modification (Supplementary Figure S1b). The determined structural characteristics of the carboxymethylated derivative of ScF, ScFCM, used in this study are presented in Table 1.

The Effect of Fucoidan from S. cichorioides and Its Carboxymethylated Derivative on the Glucose Uptake and the Lactate and Glutamate Production in Human Melanoma Cells
In this study, we determined the metabolic profile of normal human immortal keratinocyte cells (HaCaT) and human melanoma cells (SK-MEL-28, SK-MEL-5, and RPMI-7951) by their ability to uptake glucose induced by insulin or excrete lactate and glutamate.
We found that the HaCaT cells took up 24 pmol of 2-deoxy-glucose (2-DG) without insulin stimulation or 45 pmol of 2-DG induced by insulin, indicating a moderate ability of this cell type to take up glucose (Figure 1a). The SK-MEL-28 and RPMI-7951 cells took up 172 pmol and 123 pmol of 2-DG without insulin, respectively, while the insulin stimulation induced an increase in glucose uptake by 230 and 204 pmol, respectively, which is typical for cells of a glycolytic metabolic profile (Figure 1b,c). In contrast, SK-MEL-5 cells were able to take up 36 or 68 pmol of glucose without or with insulin stimulation, respectively ( Figure 1d).
Modification of polysaccharides is one of the promising ways of obtaining new compounds for medicine, chemistry, ecology, and industry. In the present study, the carboxymethylation of ScF was based on the reaction of the O-alkylation of the polysaccharide with acetic acid in NaOH and isopropanol.
We studied the structure of the obtained polysaccharide, ScFCM, via 13 C NMR spectroscopy (Supplementary Figure S1b). It confirmed that this derivative contained carboxymethyl groups. Thus, the signals belonging to the carbonyl group at 178.8 ppm and to the methylene carbon at 71.1 ppm appeared in the spectrum after the modification (Supplementary Figure S1b). The determined structural characteristics of the carboxymethylated derivative of ScF, ScFCM, used in this study are presented in Table 1.

The Effect of Fucoidan from S. cichorioides and Its Carboxymethylated Derivative on the Glucose Uptake and the Lactate and Glutamate Production in Human Melanoma Cells
In this study, we determined the metabolic profile of normal human immortal keratinocyte cells (HaCaT) and human melanoma cells (SK-MEL-28, SK-MEL-5, and RPMI-7951) by their ability to uptake glucose induced by insulin or excrete lactate and glutamate.
We found that the HaCaT cells took up 24 pmol of 2-deoxy-glucose (2-DG) without insulin stimulation or 45 pmol of 2-DG induced by insulin, indicating a moderate ability of this cell type to take up glucose (Figure 1a). The SK-MEL-28 and RPMI-7951 cells took up 172 pmol and 123 pmol of 2-DG without insulin, respectively, while the insulin stimulation induced an increase in glucose uptake by 230 and 204 pmol, respectively, which is typical for cells of a glycolytic metabolic profile (Figure 1b,c). In contrast, SK-MEL-5 cells were able to take up 36 or 68 pmol of glucose without or with insulin stimulation, respectively ( Figure 1d).  , and SK-MEL-5 (d) to take up 2-DG as determined by Glucose Uptake Colorimetric Assay. The ability of SK-MEL-28 (e), RPMI-7951 (f), and SK-MEL-5 (g) cells to excrete the lactate and glutamate as determined by Lactate/Glutamate Glo assay. Data show the mean of three independent experiments ± SD. A one-way ANOVA and Tukey's HSD test for multiple comparisons indicated the statistical significance (ns-no significant difference, * p < 0.05, ** p < 0.01).
To confirm the preferred metabolic profile of the cancer cells tested, we also estimated the lactate and glutamate production. Since the lactate produced by glycolysis is released from the cell and the glutamate produced by oxidative phosphorylation is accumulated within the cell, we analyzed both cell lysates and cell culture media. As a result, the amount of lactate in the culture medium of SK-MEL-28 melanoma cells reached 13.98 × 10 6 and 21.75 × 10 6 pmol, while the amount of lactate produced by RPMI-7951 or SK-MEL-5 cells was not significant (Figure 1e-g). The amount of glutamate in the cell lysates of SK-MEL-28, RPMI-7951, and SK-MEL-5 was 3.61 × 10 6 and 3.96 × 10 6 pmol; 11.91 × 10 6 and 15.46 × 10 6 pmol; and 30.01 × 10 6 and 38.80 × 10 6 pmol at 24 and 48 h, respectively (Figure 1e-g).
The results obtained provided evidence that normal human immortal keratinocyte cells (HaCaT) are characterized by the modest ability to take up glucose and produce lactate and glutamate, which is inherent in normal cells. The glycolysis metabolic pathway with high lactate excretion is preferable only for SK-MEL-28 cells among the tested melanoma cells (Figure 1b,e). RPMI-7951 cells were able to take up a high amount of glucose but produced fewer amounts of lactate and glutamate (Figure 1c,f). SK-MEL-5 was determined to excrete a high amount of glutamate that was characteristic of cells predominantly with mitochondrial oxidative phosphorylation (OXPHOS) metabolic profile (Figure 1d,g).
In the present study, we assessed the effect of ScF from S. cichorioides and ScFCM on glucose uptake and lactate and glutamate production in SK-MEL-28 cells that have a glycolytic metabolic profile (Figure 2a,b). We found that ScF at concentrations of 50, 100, and 200 µg/mL inhibited the insulin-stimulated uptake of 2-DG in SK-MEL-28 cells by 16,22, and 34%, respectively, compared to PBS-treated cells stimulated with insulin (control) (Figure 2a). ScFCM at 50, 100, and 200 µg/mL had a more potent inhibiting effect on glucose uptake and reduced the amount of 2-DG in SK-MEL-28 cells by 21, 28, and 42%, respectively, compared to the control (Figure 2a). ScF (at 50, 100, and 200 µg/mL) had a slight effect on the lactate and glutamate production in SK-MEL-28 cells; the percentage of inhibition was less than 15% compared to the control (Figure 2b). ScFCM (at 50, 100, and 200 µg/mL) decreased the lactate content by 11, 14, and 20%, respectively, and the glutamate production in SK-MEL-28 cells by 14,22, and 28%, respectively ( Figure 2b). Thus, ScF and ScFCM were shown to possess a comparable inhibiting effect on glucose uptake and slightly influenced the lactate/glutamate production in SK-MEL-28 cells.
To confirm the preferred metabolic profile of the cancer cells tested, we also estimated the lactate and glutamate production. Since the lactate produced by glycolysis is released from the cell and the glutamate produced by oxidative phosphorylation is accumulated within the cell, we analyzed both cell lysates and cell culture media. As a result, the amount of lactate in the culture medium of SK-MEL-28 melanoma cells reached 13.98 × 10 6 and 21.75 × 10 6 pmol, while the amount of lactate produced by RPMI-7951 or SK-MEL-5 cells was not significant (Figure 1e-g). The amount of glutamate in the cell lysates of SK-MEL-28, RPMI-7951, and SK-MEL-5 was 3.61 × 10 6 and 3.96 × 10 6 pmol; 11.91 × 10 6 and 15.46 × 10 6 pmol; and 30.01 × 10 6 and 38.80 × 10 6 pmol at 24 and 48 h, respectively (Figure 1e-g).
The results obtained provided evidence that normal human immortal keratinocyte cells (HaCaT) are characterized by the modest ability to take up glucose and produce lactate and glutamate, which is inherent in normal cells. The glycolysis metabolic pathway with high lactate excretion is preferable only for SK-MEL-28 cells among the tested melanoma cells (Figure 1b,e). RPMI-7951 cells were able to take up a high amount of glucose but produced fewer amounts of lactate and glutamate (Figure 1c,f). SK-MEL-5 was determined to excrete a high amount of glutamate that was characteristic of cells predominantly with mitochondrial oxidative phosphorylation (OXPHOS) metabolic profile (Figure 1d,g).
In the present study, we assessed the effect of ScF from S. cichorioides and ScFCM on glucose uptake and lactate and glutamate production in SK-MEL-28 cells that have a glycolytic metabolic profile (Figure 2a,b). We found that ScF at concentrations of 50, 100, and 200 µg/mL inhibited the insulin-stimulated uptake of 2-DG in SK-MEL-28 cells by 16,22, and 34%, respectively, compared to PBS-treated cells stimulated with insulin (control) (Figure 2a). ScFCM at 50, 100, and 200 µg/mL had a more potent inhibiting effect on glucose uptake and reduced the amount of 2-DG in SK-MEL-28 cells by 21, 28, and 42%, respectively, compared to the control (Figure 2a). ScF (at 50, 100, and 200 µg/mL) had a slight effect on the lactate and glutamate production in SK-MEL-28 cells; the percentage of inhibition was less than 15% compared to the control (Figure 2b). ScFCM (at 50, 100, and 200 µg/mL) decreased the lactate content by 11, 14, and 20%, respectively, and the glutamate production in SK-MEL-28 cells by 14,22, and 28%, respectively ( Figure 2b). Thus, ScF and ScFCM were shown to possess a comparable inhibiting effect on glucose uptake and slightly influenced the lactate/glutamate production in SK-MEL-28 cells.  Figure 2. The effect of fucoidan and its carboxymethylated derivative on glucose uptake and lactate and glutamate production in SK-MEL-28 melanoma cells. The effect of fucoidan from S. cichorioides (ScF) and its carboxymethylated derivative (ScFCM) on (a) the insulin-stimulated uptake of glucose and (b) the lactate and glutamate production. Data show the mean of three independent experiments ± SD. A one-way ANOVA and Tukey's HSD test for multiple comparisons indicated the statistical significance (ns-no significant difference, * p < 0.05, ** p < 0.01).

The Metabolically Oriented Effect of Fucoidan from S. cichorioides and Its Carboxymethylated Derivative on Viability and Proliferation of Human Melanoma Cells
In our study, we tested the idea that the fucoidan from the brown alga S. cichorioides or its carboxymethylated derivative in combination with a low dose of 2-DG would synergistically delay the glycolysis of SK-MEL-28 melanoma cells to result in pronounced inhibition of cells' viability and proliferation.
At the first stage of bioactivity investigations, we assessed the individual effect of 2-DG (0.1-20 mM), ScF (100-800 µg/mL), and ScFCM (100-800 µg/mL) on the viability and proliferation of SK-MEL-28 cells in order to calculate their half-maximal inhibitory concentration (IC 50 ) and select the effective concentrations for combinatorial treatment.
The IC 50 for SK-MEL-28 cells treated with an inhibitor of glycolysis 2-DG was estimated at 8.7 mM after 72 h of cells incubation (Figure 3a). The IC 50 for fucoidan ScF was not distinguished at concentrations up to 800 µg/mL. ScF at 100, 200, 400, and 800 µg/mL inhibited the viability of SK-MEL-28 cells by 5, 12, 17, and 24%, respectively, at 72 h of cell incubation (Figure 3b (ScF) and its carboxymethylated derivative (ScFCM) on (a) the insulin-stimulated uptake of glucose and (b) the lactate and glutamate production. Data show the mean of three independent experiments ± SD. A one-way ANOVA and Tukey's HSD test for multiple comparisons indicated the statistical significance (ns-no significant difference, * p < 0.05, ** p < 0.01).

The Metabolically Oriented Effect of Fucoidan from S. cichorioides and Its Carboxymethylated Derivative on Viability and Proliferation of Human Melanoma Cells
In our study, we tested the idea that the fucoidan from the brown alga S. cichorioides or its carboxymethylated derivative in combination with a low dose of 2-DG would synergistically delay the glycolysis of SK-MEL-28 melanoma cells to result in pronounced inhibition of cells' viability and proliferation.
At the first stage of bioactivity investigations, we assessed the individual effect of 2-DG (0.1-20 mM), ScF (100-800 µg/mL), and ScFCM (100-800 µg/mL) on the viability and proliferation of SK-MEL-28 cells in order to calculate their half-maximal inhibitory concentration (IC50) and select the effective concentrations for combinatorial treatment.
The  As shown in Figure 4a,b, 2-DG at a concentration of 1 mM inhibited the viability of SK-MEL-28 cells by 20% compared to non-treated cells (control). As shown in Figure 4a,b, 2-DG at a concentration of 1 mM inhibited the viability of SK-MEL-28 cells by 20% compared to non-treated cells (control).

The Metabolically Oriented Effect of Fucoidan from S. cichorioides and Its Carboxymethylated Derivative on the Colony Formation of Human Melanoma Cells
Then we tested whether the fucoidan from S. cichorioides (ScF) or its carboxymethylated derivative (ScFCM) increased the inhibiting activity of 2-DG on the colony formation in human melanoma cells (SK-MEL-28) using the soft agar assay. First, we measured the effect of each of the investigated compounds separately (Figure 5a

The Metabolically Oriented Effect of Fucoidan from S. cichorioides and Its Carboxymethylated Derivative on the Colony Formation of Human Melanoma Cells
Then we tested whether the fucoidan from S. cichorioides (ScF) or its carboxymethylated derivative (ScFCM) increased the inhibiting activity of 2-DG on the colony formation in human melanoma cells (SK-MEL-28) using the soft agar assay. First, we measured the effect of each of the investigated compounds separately (Figure 5a

The Molecular Mechanism of the Metabolically Oriented Effect of Fucoidan from S. cichorioides and Its Carboxymethylated Derivative
In recent decades, research efforts have focused on targeting cancer cell metabolism since it strongly differs from glucose metabolism in normal cells [26]. In this study, we assessed the effect of an inhibitor of glycolysis, 2-DG, in combination with ScF or ScFCM on the activity of members of glycolysis by Western blot analysis (Figure 7a,b). The investigated compounds alone slightly influenced the expression of the glucose transporter Glut 1, while the combined treatment of SK-MEL-28 cells with 2-DG (1 mM) and ScF (100 and 200 µg/mL) or ScFCM (100 and 200 µg/mL) led to its complete down-regulation ( Figure  7a,b). The expression of Hexokinase II, glyceraldehyde-3-phosphate-dehydrogenase (GAPDH), and pyruvate kinase M2 (PKM2) proteins seemed to be unaltered after treatment with the polysaccharides ScF and ScFCM (200 µg/mL) separately; however, ScF or ScFCM at 100 and 200 µg/mL enhanced the

The Molecular Mechanism of the Metabolically Oriented Effect of Fucoidan from S. cichorioides and Its Carboxymethylated Derivative
In recent decades, research efforts have focused on targeting cancer cell metabolism since it strongly differs from glucose metabolism in normal cells [26]. In this study, we assessed the effect of an inhibitor of glycolysis, 2-DG, in combination with ScF or ScFCM on the activity of members of glycolysis by Western blot analysis (Figure 7a,b). The investigated compounds alone slightly influenced the expression of the glucose transporter Glut 1, while the combined treatment of SK-MEL-28 cells with 2-DG (1 mM) and ScF (100 and 200 µg/mL) or ScFCM (100 and 200 µg/mL) led to its complete down-regulation (Figure 7a,b). The expression of Hexokinase II, glyceraldehyde-3-phosphate-dehydrogenase (GAPDH), and pyruvate kinase M2 (PKM2) proteins seemed to be unaltered after treatment with the polysaccharides ScF and ScFCM (200 µg/mL) separately; however, ScF or ScFCM at 100 and 200 µg/mL enhanced the inhibiting effect of 2-DG (1 mM) on Hexokinase II and PKM2 activation. The GAPDH expression did not change under the same experimental conditions. The expression levels of lactate dehydrogenase A (LDHA) and pyruvate dehydrogenase, which control the pyruvate to lactate conversion and play a key role in glycolysis, cell growth, and tumor maintenance, proved to be significantly suppressed by combined treatment with 2-DG and ScF or ScFCM (Figure 7a,b), which led to the disruption of the metabolic pathway of SK-MEL-28 melanoma cells and the suppression of cell proliferation. of lactate dehydrogenase A (LDHA) and pyruvate dehydrogenase, which control the pyruvate to lactate conversion and play a key role in glycolysis, cell growth, and tumor maintenance, proved to be significantly suppressed by combined treatment with 2-DG and ScF or ScFCM (Figure 7a,b), which led to the disruption of the metabolic pathway of SK-MEL-28 melanoma cells and the suppression of cell proliferation.

Discussion
Melanoma is a highly aggressive skin cancer with an increasing incidence, and the development of novel treatment strategies remains an urgent need [27]. Cancer cells that are phenotypically characterized by aerobic glycolysis prefer glucose uptake and lactate production, even in the presence of oxygen (Warburg effect), while glutamine is extremely important for oxidative phosphorylation (OXPHOS) and redox regulation [9,28,29]. Metabolic reprogramming (plasticity) in cancer is rarely static but, instead, a highly dynamic process that allows rapid adaptability, which requires both the flexibility to utilize different metabolic substrates and the ability to process metabolic substrates in different ways. It has been shown that metabolic reprogramming is a key driver of melanoma progression and response to current standard-of-care anticancer and immune therapies [30]. The inherent plasticity of melanoma cell metabolism has been evidenced by reversible metabolome alterations that occur during metastasis and response to anticancer therapies and in the diversity of fuel sources melanoma cells can utilize to survive in response to nutrient deprivation and exposure to different microenvironmental niches [9,31,32]. As such, this inherent metabolic plasticity creates a moving target for therapeutic interventions and consequently poses a major challenge to effective therapy.
The sulfated polysaccharides of brown algae, fucoidans, and their derivatives proved to exhibit potent anti-proliferative, anti-migratory, anti-metastatic, and metabolically oriented activities against human cancer cells and could be used as promising metabolic modifiers for increasing the effectiveness of melanoma therapy [16,33,34]. In the present study, we identified the fucoidan isolated from the brown alga S. cichorioides as almost pure fucan, consisting of a backbone of 1,3-linked α-L-fucopyranose residues with a small proportion of 1,4-linked α-L-fucopyranose residues. A small amount of single α-L-fucose residues were present in the branches at position 2. Sulfate groups occupied positions 2 and 4 of the fucopyranose residues. The fucoidan from S. cichorioides was carboxymethylated in order to improve its functional Relative band density was measured using the Quantity One 1D analysis software version 4.6.7 (Bio-Rad, Hercules, CA, USA). Band density was normalized to β-actin total level. Results are presented as mean ± standard deviation (SD); * p < 0.05, ** p < 0.01.

Discussion
Melanoma is a highly aggressive skin cancer with an increasing incidence, and the development of novel treatment strategies remains an urgent need [27]. Cancer cells that are phenotypically characterized by aerobic glycolysis prefer glucose uptake and lactate production, even in the presence of oxygen (Warburg effect), while glutamine is extremely important for oxidative phosphorylation (OXPHOS) and redox regulation [9,28,29]. Metabolic reprogramming (plasticity) in cancer is rarely static but, instead, a highly dynamic process that allows rapid adaptability, which requires both the flexibility to utilize different metabolic substrates and the ability to process metabolic substrates in different ways. It has been shown that metabolic reprogramming is a key driver of melanoma progression and response to current standard-of-care anticancer and immune therapies [30]. The inherent plasticity of melanoma cell metabolism has been evidenced by reversible metabolome alterations that occur during metastasis and response to anticancer therapies and in the diversity of fuel sources melanoma cells can utilize to survive in response to nutrient deprivation and exposure to different microenvironmental niches [9,31,32]. As such, this inherent metabolic plasticity creates a moving target for therapeutic interventions and consequently poses a major challenge to effective therapy.
The sulfated polysaccharides of brown algae, fucoidans, and their derivatives proved to exhibit potent anti-proliferative, anti-migratory, anti-metastatic, and metabolically oriented activities against human cancer cells and could be used as promising metabolic modifiers for increasing the effectiveness of melanoma therapy [16,33,34]. In the present study, we identified the fucoidan isolated from the brown alga S. cichorioides as almost pure fucan, consisting of a backbone of 1,3-linked α-L-fucopyranose residues with a small proportion of 1,4-linked α-L-fucopyranose residues. A small amount of single α-L-fucose residues were present in the branches at position 2. Sulfate groups occupied positions 2 and 4 of the fucopyranose residues. The fucoidan from S. cichorioides was carboxymethylated in order to improve its functional properties. Several studies have demonstrated that the chemical modifications of polysaccharides, such as sulfation, amination, phosphorylation, and selenization, significantly influence their structural composition, molecular weight, linkage pattern, and ionic characteristics of polysaccharides; as a result, the functional properties of polysaccharides are also modified [21,22,35,36].
We have found that the human melanoma cells tested (SK-MEL-28, RPMI-7951, and SK-MEL-5) differed in the ability to take up glucose and excrete the lactate and glutamate that might be related to the metabolic plasticity of cell lines even within one cancer type (Figure 1). SK-MEL-28 cells were determined to take up glucose and excrete lactate at a high rate but did not show the glutamate production characteristic of aerobic glycolysis (Figure 1b,e). RPMI-7951 cells were able to take up a high amount of glucose but produced smaller amounts of lactate and glutamate, and SK-MEL-5 cells were characterized by a low glucose uptake and lactate excretion but a high glutamate production (Figure 1c,d,f,g). In our work, we focused on the SK-MEL-28 melanoma cell line having aerobic glycolysis as a preferable metabolic pathway.
Thus, we have found for the first time that the fucoidan from S. cichorioides and its carboxymethylated derivative can decrease glucose uptake and lactate excretion in SK-MEL-28 melanoma cells in a dose-dependent manner ( Figure 2). Previously, the effect of fucoidans from brown algae on glucose uptake was investigated in order to assess their anti-diabetic potential. It was demonstrated that the treatment with fucoidan from brown alga Undaria pinnatifida stimulated glucose uptake in normal 3T3 adipocytes and restored insulin-stimulated glucose uptake in obesity-induced insulin-resistant adipocytes [17]. Shan and co-authors [37] indicated the potential effects of fucoidan from Ascophyllum nodosum on the regulation of blood glucose levels by direct inhibition of glucose transport via SGLT1, causing the glucose transport to markedly reduce and relieving postprandial hyperglycemia.
2-Deoxy-D-glucose (2-DG), a glucose analog inhibiting glycolysis, is widely used as a metabolic modifier to disturb cancer cell proliferation [12]. However, the potential for using 2-DG as a single therapeutic agent appears to be limited by the development of toxicity, diaphoresis, hypoglycemia, and disturbances of the CNS through daily administrations of large doses of 2-DG over a long period of time [38]. Thus, we hypothesized that a combination of 2-DG and fucoidan or its carboxymethylated derivative could enhance the inhibition of melanoma cell proliferation at lower, non-toxic concentrations, thereby minimizing probable side effects of 2-DG and potentially helping to overcome the therapeutic resistance of melanoma cells.
2-DG, sulfated 1,3-linked α-L-fucan from brown alga S. cichorioides (ScF), and its carboxymethylated derivative (ScFCM) were separately shown to inhibit the viability, proliferation, and colony formation of melanoma cells to variable degrees (Figures 3 and 4). Recently, it has been reported that 2-DG inhibited the viability and proliferation of human cancer cells via induction of apoptosis [11,39,40]. Our results on the anticancer activity of fucoidan from S. cichorioides are also consistent with the published data, which indicate that it significantly inhibited TPA-induced neoplastic transformation of mouse epidermal cells JB6 Cl41 [24,41]. In our study, we, for the first time, assessed the effect of the carboxymethylated derivative of ScFCM on the viability, proliferation, and colony formation of SK-MEL-28 cells. The anticancer activity of ScFCM was comparatively higher than that of the native polysaccharide, which can be explained by its increased molecular weight, solubility in water, decreased viscosity, and a change in the molar ratios of monosaccharides.
The results obtained in this study indicate that the fucoidan from the brown alga S. cichorioides and its carboxymethylated derivative at low, non-toxic concentrations have a synergistic metabolically-oriented effect in combination with 2-DG that causes a pronounced inhibition of the viability, proliferation, and colony formation of SK-MEL-28 melanoma cells (Figures 4-6). In the recent decade, the combined effect of 2-DG with chemotherapeutic drugs has been intensively investigated in order to increase the efficiency of anticancer therapy. Thus, the combination of 2-DG and cisplatin was shown to enhance the cytotoxicity in human head and neck cancer FaDu cells by mechanisms involving oxidative stress [42]. Also, 2-DG in combination with fenofibrate, safely used for over 40 years to decrease blood cholesterol in patients, synergistically inhibited the viability of human melanoma NM2C5, osteosarcoma 143B, and breast adenocarcinoma SKBR3 cell lines through regulation of activities of the AMPK and mTOR proteins, which led to greater ER stress and apoptosis induction [43]. Additionally, it was found that the combined treatment with 2-DG and sorafenib, an inhibitor of tyrosine protein kinases such as VEGFR, PDGFR, and the Raf family kinases, increased apoptosis and inhibited colony formation of the hepatocellular carcinoma cell line Hep3B and Huh7 cells [44]. To the best of our knowledge, the present study is the first to emphasize the potential of the combination of 2-DG with fucoidan and its carboxymethylated derivative as an approach to cancer treatment.
Glycolysis is a metabolic pathway that converts glucose into pyruvate in the cytoplasm, which leads to the production of adenosine triphosphate (ATP). The entire pathway of glycolysis contains ten steps of chemical reactions, with each catalyzed by a specific enzyme [45]. After glucose is taken up by membrane glucose transporters (Glut), which are overexpressed in cancer cells, it is converted into glucose-6-phosphate by hexokinase II. Then phosphofructokinase (PFK) catalyzes the phosphorylation of fructose-6-phosphate into fructose-1,6-bisphosphate. Glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) then converts glyceraldehyde-3-phosphate into 1,3-bisphosphoglycerate. The enzyme pyruvate kinase M2 (PKM2) catalyzes the irreversible phosphoryl group transfer from phosphoenolpyruvate to pyruvate, from which ATP is formed. Cancer cells switch to and depend on aerobic glycolysis for survival. Therefore, lactate dehydrogenase (LDH), which catalyzes the conversion of pyruvate to lactate, is the key enzyme for determining the glycolytic phenotype of cancer cells; as such, it could be utilized as a therapeutic target. The pyruvate dehydrogenase complex catalyzes the conversion of pyruvate and CoA into acetyl-CoA and CO 2 in the presence of NAD + [46]. It has been reported that 2-DG competes with glucose for transport into the cell and can competitively inhibit glucose transport. The expression of glucose transporters and glycolytic enzymes increases under hypoxia, which is characteristic of cancer cells; this, in turn, enhances the uptake of 2-DG by cancer cells compared to normal cells under aerobic conditions [47][48][49]. In our study, 2-DG, fucoidan (ScF), and its carboxymethylated derivative (ScFCM) separately influenced slightly the expression of the Glut 1 transporter at low, non-toxic concentrations, but the treatment of SK-MEL-28 cells with 2-DG in combination with ScF or ScFCM further enhanced the inhibitory effect (Figure 7).
After entering the cell, 2-DG is phosphorylated by Hexokinase II to 2-deoxy-D-glucose-6-phosphate (2-DG-6-P), but, unlike glucose, 2-DG-6-P cannot be further metabolized by phosphoglucose isomerase (PGI) into a 5-carbon ring [12]. The results obtained in our study show that the combination of 2-DG with fucoidan or its derivative down-regulate the activities of Hexokinase II, PKM2, LDHA, and pyruvate dehydrogenase, which are the major components of the glycolysis pathway (Figure 7). This leads to the inhibition of growth colony formation and, consequently, the death of SK-MEL-28 melanoma cells.
To conclude, our study has assessed the feasibility and efficacy of a combined application of the glycolysis inhibitor 2-DG and a natural sulfated polysaccharide from the brown alga S. cichorioides and its derivatives to treat the aggressive human melanoma cells SK-MEL-28 and inhibit their proliferation associated with high glycolysis. Further investigations of the metabolically oriented effect of the combination of 2-DG with fucoidan or its derivative in vivo are of particular scientific interest. This combined strategy, due to the effective inhibition of uncontrolled cell proliferation and the enhancement of the therapeutic effect, is expected to be used in melanoma therapy in the future.

Reagents
The organic solvents, inorganic salts, and acids used in the study were manufactured by Dia-m (Moscow, Russia). The sorbent for chromatography Macro-Prep DEAE was purchased from Bio Rad Laboratories, Inc. (Hercules, CA, USA). The phosphate-buffered saline (PBS), L-glutamine, penicillin/streptomycin solution (10,000 U/mL, 10 µg/mL), Minimum Essential Medium Eagle (MEM), and reference standards (mannose, rhamnose, glucose, galactose, xylose, and dextrans) were purchased from the Sigma-Aldrich company

Isolation and Chemical Modification of Fucoidan from the Brown Alga S. cichorioides 4.2.1. Brown Alga
The sample of the brown alga S. cichorioides (Sc) was collected in August 2021 from Peter the Great Bay, Sea of Japan (Russia). Fresh algal biomass was powdered and pretreated with 70% aqueous ethanol (=1:10 w/v) at room temperature for 10 days. The defatted alga was air-dried.

Isolation of Fucoidan from the Brown Alga S. cichorioides
The fucoidan (ScF) was isolated from the brown alga sample (100 g) via the earlier described method [24] with a yield of 4 g.

Carboxymethylation of Fucoidan from the Brown Alga S. cichorioides
Carboxymethylation of ScF was performed via the previously described method with some modifications [50]. In brief, ScF (500 mg) was mixed with 21 mL isopropanol and stirred for 15 min at room temperature. Then the polysaccharide mixture was supplemented with 8 mL of 20% NaOH drop-wise and stirred at room temperature for 3 h. The carboxymethylated agent (4.4 g chloroacetic acid, 8 mL of 20% NaOH, and 21 mL isopropanol) was added under stirring at 60 • C for 4 h. The obtained solution was cooled to room temperature; the pH was adjusted to 7.0 with 0.5 M HCl. The product was dialyzed against H 2 O for 72 h. The sample was freeze-dried to obtain the carboxymethylated derivative of fucoidan (hereinafter referred to as ScFCM).

Content of Carbohydrates
The content of carbohydrates was determined via the method of Michel Dubois et al. [51]. The absorbance was measured at 490 nm on a Power Wave XS microplate reader (BioTek, Winooski, VT, USA). Glucose (1 mg/mL) was used as the reference standard.

Content of Sulfate Groups
The content of sulfate groups was determined via the turbidimetric method after hydrolysis of the sulfated fucoidan and its derivative with 1N HCl [52]. The absorbance was measured at 360 nm on a Power Wave XS microplate reader (BioTek, Winooski, VT, USA). K 2 SO 4 (1 mg/mL) was used as the reference standard.

Monosaccharide Composition
The monosaccharide composition of ScF and ScFCM was determined via gas-liquid chromatography (GLC) after hydrolysis using 2 M TFA (6 h, 100 • C) and obtainment of alditol acetate derivatives.

Molecular Weight
The molecular weight of the ScF and ScFCM was determined via size-exclusion chromatography (SEC) on an Agilent 1100 Series HPLC system (Agilent Technologies, Waldbronn, Germany) equipped with a refractive index detector and series-connected SEC columns, Shodex OHpak SB-805 HQ and OHpak SB-803 HQ (Showa Denko, Tokyo, Japan). Cells were cultured in MEM supplemented with 10% heat-inactivated FBS and 1% penicillin/streptomycin solution at 37 • C in a humidified atmosphere containing 5% CO 2 . The number of passages was carefully controlled, and mycoplasma contamination was monitored on a regular basis.

Glucose Uptake Assay
The HaCaT cells (5.0 × 10 4 /mL) and SK-MEL-28, RPMI-7951, and SK-MEL-5 cells (2.0 × 10 4 /mL) were seeded in a 24-well plate and cultured at 37 • C in a 5% CO 2 incubator for 24 h. Then the cells were treated with either PBS (vehicle control) or ScF or ScFCM at concentrations of 50, 100, and 200 µg/mL for 24 h. The cells were then washed twice with PBS and starved in 1 mL of serum-free medium overnight. Afterward, they were washed thrice with PBS, glucose-starved by plating with 1 mL of the Krebs-Ringer-Phosphate-HEPES (KRPH) buffer (20 mM HEPES, 5 mM KH 2 PO 4 , 1 mM MgSO 4 , 1 mM CaCl 2 , 136 mM NaCl, and 4.7 mM KCl, pH 7.4, 2% BSA) for 40 min, and then stimulated with insulin (1 mM) for 20 min. Then 2-deoxy-D-glucose (2-DG) (10 mM) was added, and the cells were incubated for an additional 20 min. The cells were lysed by extraction buffer, the cell lysate was collected from the wells, and glucose uptake was measured using a Glucose Uptake Colorimetric Assay Kit (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer's instructions. The absorbance was measured at 412 nm on a Power Wave XS microplate reader (BioTek, Winooski, VT, USA) maintained at 37 • C. 2-Deoxy-D-glucose 6-phosphate (2-DG6P) (0.01 mM) was used as the reference standard.

Lactate and Glutamate Production Assay
The SK-MEL-28, RPMI-7951, and SK-MEL-5 cells (2.5 × 10 4 /mL) were seeded in a 96-well plate and cultured at 37 • C in a 5% CO 2 incubator for 24 h. Then the cells were treated with PBS or ScF at concentrations of 50, 100, and 200 µg/mL for 48 h. The culture medium was collected and used to determine the level of lactate production by the Lactate-Glo TM Assay (Promega, Fitchburg, WI, USA) according to the manufacturer's protocol. The cells remaining attached were washed twice with PBS and used to determine the level of glutamate production by the Glutamate-Glo TM Assay (Promega, Fitchburg, WI, USA) according to the manufacturer's protocol. The level of lactate and glutamate content was determined by the luminescence method on a PHERAstar FS multi-mode microplate reader (BMG Labtech, Offenburg, Germany) using the appropriate calibration curves.

Western Blot Assay
The SK-MEL-28 cells (1.0 × 10 5 /mL) were seeded in 100 mm dishes and incubated for 24 h at 37 • C in a CO 2 incubator. The cells were treated with 2-DG (1 mM) for 24 h and then with ScF or ScFCM at 100 and 200 µg/mL for an additional 48 h. The preparations of the cell lysate and the Western blot assay were previously described in the study by Vishchuk et al. [53]. In brief, the protein content was determined by the DC protein assay (Bio-Rad, Hercules, CA, USA). Lysates of protein (20-40 µg) were exposed to 10% or 12% SDS-PAGE and electrophoretically transferred to polyvinylidene difluoride membranes (PVDF) (Millipore, Burlington, MA, USA). The membranes were blocked with 5% non-fat milk (Bio-Rad) for 1 h and then incubated with the respective specific primary antibody at 4 • C overnight. Protein bands were visualized using an enhanced chemiluminescence reagent (ECL) (Bio-Rad, Hercules, CA, USA) after hybridization with an HRP-conjugated secondary antibody. The band density was quantified using the Quantity One 1D analysis software, version 4.6.7 (Bio-Rad).

Statistical Analysis
All the assays were performed in at least triplicate. The results were expressed as mean ± standard deviation (SD). The obtained data were statistically processed by the one-way analysis of variance (ANOVA) and Tukey's HSD test with significance levels of * p < 0.05 and ** p < 0.01.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.