Combined Anticancer Effect of Sulfated Laminaran from the Brown Alga Alaria angusta and Polyhydroxysteroid Glycosides from the Starfish Protoreaster lincki on 3D Colorectal Carcinoma HCT 116 Cell Line

Colorectal cancer is one of the most frequent types of malignancy in the world. The search for new approaches of increasing the efficacy of cancer therapy is relevant. This work was aimed to study individual, combined anticancer effects, and molecular mechanism of action of sulfated laminaran AaLs of the brown alga Alaria angusta and protolinckiosides A (PL1), B (PL2), and linckoside L1 (L1) of the starfish Protoreaster lincki using a 3D cell culture model. The 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS), soft agar, 3D spheroids invasion, and Western blotting assays were performed to determine the effect and mechanism of the action of investigated compounds or their combinations on proliferation, colony formation, and the invasion of 3D HCT 116 spheroids. AaLs, PL1, PL2, and L1 individually inhibited viability, colony growth, and the invasion of 3D HCT 116 spheroids in a variable degree with greater activity of linckoside L1. AaLs in combination with L1 exerted synergism of a combined anticancer effect through the inactivation of protein kinase B (AKT) kinase and, consequently, the induction of apoptosis via the regulation of proapoptotic/antiapoptotic proteins balance. The obtained data about the efficacy of the combined anticancer effect of a laminaran derivative of brown algae and polyhydroxysteroid glycosides of starfish open up prospects for the development of new therapeutic approaches for colorectal cancer treatment.


Introduction
Oncological diseases are one of the main causes of human mortality in the modern world, and the economic and financial burden for cancer research is increasing [1]. Colorectal cancer is the most frequently diagnosed malignant cancer around the world, and one of the most common causes of death among cancer patients [2]. Approximately 20 to 50% of patients already have distant metastases at the time of diagnosis [3]. Unfortunately, many radically operated on patients experience dissemination of the process at different periods after the operation, and the overall 5-year survival rate of patients with metastatic colon cancer does not exceed 7-8% [4]. Despite the noticeable progress in the treatment of colorectal cancer using cytotoxic and targeted drugs, the effectiveness of chemotherapy for this type of cancer cannot be considered satisfactory, which dictates the need to search for new approaches in the treatment of patients with advanced colorectal cancer [5]. For instance, the combination of two or more therapeutic treatments specifically regulating

Isolation, Modification of Laminaran from A. angusta, and Structural Characteristics of Sulfated Laminaran
Laminaran from the brown alga A. angusta was isolated according to a modified method described in recent work [22]. To remove low molecular weight compounds, the alga was treated with organic solvents and air-dried. Then the extraction was performed with 0.1 N hydrochloric acid at 60 • C for 2 h, dialyzed, concentrated, precipitated with four volumes of 96% ethanol, and air-dried in order to obtain crude fractions of watersoluble polysaccharides (laminarans and fucoidans). A highly purified preparation of laminaran was obtained during anionexchange chromatography on a Macro-prep DEAE and hydrophobic chromatography on polychrome. The structural characteristics of native laminaran AaL from A. angusta were determined using high-performance liquid chromatography (HPLC), chemical (methods for determining the content of total carbohydrates and sulfates), and 13 C NMR spectroscopy (Table 1, Supplementary Figure S1A). It was confirmed that laminaran AaL has a backbone of 1,3-linked β-D-glucopyranose residues with single glucose branches at C-6 with a bond ratio of 1,3:1,6 = 6:1 (Supplementary Figure  S1A). The laminarans with similar structural characteristics were isolated from the brown algae Dictyota dichotoma (order Dictyotales) [23], Alaria marginata [22], Saccharina cichorioides and from Saccharina japonica [14], Saccharina gurjanovae [24] (order Laminariales), Coccophora langsdorfii [25], Sargassum duplicatum [26], and Sargassum fusiforme [27] (order Fucales). To enhance the anticancer activity of the native laminaran from A. angusta, it was subjected to a chemical modification. The sulfated laminaran was obtained as a result of the reaction of nucleophilic substitution of hydroxyl groups of the molecules of laminaran from A. angusta by sulfate groups using a chlorosulfonic-pyridine complex as a sulfating agent. The yield of obtained sulfated laminaran AaLs was 89%. The structural characteristics of sulfated laminaran AaLs are presented in Table 1. The content of the sulfate groups of laminaran's derivative was determined by the turbidimetrical method. The sulfated laminaran AaLs from the brown alga A. angusta was sulfated by 44% ( Table 1).

The Polyhydroxysteroid Glycosides from the Starfish P. lincki
Polyhydroxysteroid glycosides PL1, PL2, and L1 were previously isolated from the starfish P. lincki according to the scheme described earlier [21].

The Polyhydroxysteroid Glycosides from the Starfish P. lincki
Polyhydroxysteroid glycosides PL1, PL2, and L1 were previously isolated from the starfish P. lincki according to the scheme described earlier [21].

The Individual and Combined Effects of Sulfated Laminaran and Polyxydroxysteroid Glycosides on Cell Viability and Proliferation of 3D HCT 116 Spheroids
Currently, the two-dimensional cell culture (2D culture) is a widely used in vitro model in cell biology. The success of this model is due to the property of most types of animal and human cells to proliferate in special culture media and form homogeneous colonies. However, the cell growth of a monolayer culture in many parameters does not reflect the true picture of tumor growth in a living organism, where interactions not only between the cells of the tumor, but also with the surrounding extracellular matrix, represented in its core by connective tissue cells and collagen cells, are of great importance in its progression. The three-dimensional cell culture, represented by

The Individual and Combined Effects of Sulfated Laminaran and Polyxydroxysteroid Glycosides on Cell Viability and Proliferation of 3D HCT 116 Spheroids
Currently, the two-dimensional cell culture (2D culture) is a widely used in vitro model in cell biology. The success of this model is due to the property of most types of animal and human cells to proliferate in special culture media and form homogeneous colonies. However, the cell growth of a monolayer culture in many parameters does not reflect the true picture of tumor growth in a living organism, where interactions not only between the cells of the tumor, but also with the surrounding extracellular matrix, represented in its core by connective tissue cells and collagen cells, are of great importance in its progression. The three-dimensional cell culture, represented by spheroids (3D spheroids), is the most effective system that is as close as possible in properties and organization to a tumor in organisms, which is currently used for screening potential anticancer drugs [28].
The cytostatic activity of sulfated derivative of laminaran AaLs and polyhydroxysteroid glycosides PL1, PL2, and L1 was studied using a 3D cell culture model of human colorectal carcinoma cells HCT 116 (3D HCT 116 spheroids) by 3-(4, 5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. PL1 at concentrations of 6.25, 12.5, 25, 50, 100, and 200 µM was found to reduce the cell viability by 17%, 19%, 15%, 18%, 31%, and 32%, respectively, but the concentration of compounds causing 50% inhibition of the spheroids' viability (IC 50 ) was not achieved in the concentration range up to 200 µM even in 3 days of treatment ( Figure 2A). It was shown that PL2 has a comparable cytostatic effect as PL1 and insignificantly affected the viability of 3D HCT 116 spheroids at concentrations up to 200 µM ( Figure 2B). The IC 50 of linckoside L1 was determined to be 111.4, 100.0, and 89.3 µM after the 3D HCT 116 spheroids' treatment for 24 h, 48 h, and 72 h, respectively ( Figure 2C). It was found previously that compounds PL1 and L1 possessed cytotoxic activity against RAW 264.7 murine macrophage cells [21]. PL1 and L1 at a concentration of 100 µM reduced the cell viability by 20% and 50%, respectively, in 24 h of incubation. At the same time, polyhydroxysteroid glycoside PL2 did not demonstrate significant cytotoxic effects within a concentration range of 0.001-100.0 µM [21].  The sulfated laminaran's derivative AaLs exhibited a moderate antiproliferative effect against 3D HCT 116 spheroids growth at a concentration range from 400 to 1600 μg/mL with IC50 value of 1555.6, 746.7, and 469.6 μg/mL after the incubation of the spheroids for 24 h, 48 h, and 72 h, respectively ( Figure 2D). Several scientific groups have studied the effect of laminaran from the brown alga Laminaria digitata (commercially available from Sigma Aldrich (Saint Louis, MO, USA)) on the proliferation of different types of human cancer cells. Thus, it was shown that laminaran (5 mg/mL) from the brown alga L. digitata inhibited the proliferation of colorectal cancer cells HT-29 by 40% without affecting the growth of normal intestinal epithelial cells EC-6 [11]. The molecular mechanism of the antiproliferative action of laminaran was found to be associated with the regulation of members of the Fas and IGF-IR signaling pathways and the inducing of intrinsic apoptotic and ErbB pathways involved in carcinogenesis and neurodegenerative disorders [11]. It was also reported that sulfated laminaran, obtained by the chemical modification of laminaran from L. digitata, reduced the growth rate of PC-3 human prostate cells, arrested the cell cycle in the S phase, and induced apoptosis in cancer cells through the regulation of the expression level of PTEN and P271kip1 proteins [10]. Ji Y.B. and et al. showed that laminaran was able to induce the apoptosis of the human intestinal cancer cells Lovo by regulating the expression level of death receptors DR4, DR5, TRAIL, FADD, apoptotic proteins Bid, tBid, Bax, Bcl-2, and caspases 8 and 3 [12]. Additionally, the sulfated derivative of laminaran from L. digitata was obtained and its antiproliferative activity was determined. The sulfated laminaran (at The sulfated laminaran's derivative AaLs exhibited a moderate antiproliferative effect against 3D HCT 116 spheroids growth at a concentration range from 400 to 1600 µg/mL with IC 50 value of 1555.6, 746.7, and 469.6 µg/mL after the incubation of the spheroids for 24 h, 48 h, and 72 h, respectively ( Figure 2D). Several scientific groups have studied the effect of laminaran from the brown alga Laminaria digitata (commercially available from Sigma Aldrich (Saint Louis, MO, USA)) on the proliferation of different types of human cancer cells. Thus, it was shown that laminaran (5 mg/mL) from the brown alga L. digitata inhibited the proliferation of colorectal cancer cells HT-29 by 40% without affecting the growth of normal intestinal epithelial cells EC-6 [11]. The molecular mechanism of the antiproliferative action of laminaran was found to be associated with the regulation of members of the Fas and IGF-IR signaling pathways and the inducing of intrinsic apoptotic and ErbB pathways involved in carcinogenesis and neurodegenerative disorders [11]. It was also reported that sulfated laminaran, obtained by the chemical modification of laminaran from L. digitata, reduced the growth rate of PC-3 human prostate cells, arrested the cell cycle in the S phase, and induced apoptosis in cancer cells through the regulation of the expression level of PTEN and P271kip1 proteins [10]. Ji Y.B. and et al. showed that laminaran was able to induce the apoptosis of the human intestinal cancer cells Lovo by regulating the expression level of death receptors DR4, DR5, TRAIL, FADD, apoptotic proteins Bid, tBid, Bax, Bcl-2, and caspases 8 and 3 [12]. Additionally, the sulfated derivative of laminaran from L. digitata was obtained and its antiproliferative Mar. Drugs 2021, 19, 540 5 of 15 activity was determined. The sulfated laminaran (at a dose of 1.6 mg/mL) was found to have a stronger inhibitory effect on the proliferation of Lovo cancer cells than the native polysaccharide [13]. The authors suggested that the introduction of sulfate groups into the motif of laminarans changed their physicochemical properties, three-dimensional conformation and, consequently, enhanced the anticancer activity compared to native laminaran. The antitumor activity of native laminarans isolated from the brown algae S. cichorioides, S. japonica, and Fucus evanescens, as well as their sulfated derivatives, was studied by our scientific group [14]. It was confirmed that sulfated derivatives of laminarans possessed higher anticancer activity in vitro than native polysaccharides. The sulfated derivative of laminaran from F. evanescens was shown to have more pronounced anticancer activity among sulfated laminarans from S. cichorioides and S. japonica and effectively inhibited proliferation, colony formation, and migration of triple-negative breast cancer cell line MDA-MB-231 [14].
In our study, the concentrations of polyhydroxysteroid glycosides PL1, PL2, and L1 and sulfated laminaran AaLs for the combined treatment of 3D HCT 116 spheroids were chosen based on the preliminary experimental data (Supplementary Figure S2, data is represented for L1 and AaLs). So, all polyhydroxysteroid glycosides were tested in minimally inhibiting concentrations of 6.25 and 12.5 µM at which, individually, they slightly affected the viability of spheroids (the percentage of inhibition of cell viability was less than 20%) (Supplementary Figure S2). As for sulfated laminaran, based on the previous published data on the effect of the combined effect of sulfated laminaran from Dictyota dichotoma with X-ray radiation, where the radiosensitizing activity of sulfated laminaran was observed at low concentrations of 10, 20, and 40 µg/mL [16], we checked the ability of AaLs to potentiate the activity of PL1, PL2, and L1 at concentrations of 10, 20, 40, 80, and 160 µg/mL, which are much lower than its IC 50 (Supplementary Figure  S2). A strong synergism of anticancer effect was observed after the treatment of 3D HCT 116 spheroids by L1 at 12.5 µM and AaLs at 10, 20, and 40 µg/mL. The significant difference between the efficacy of the combined action of L1 (12.5 µM) and AaLs at higher concentrations of 80 and 160 µg/mL was not found compared to the action of AaLs at low concentrations of 10, 20, and 40 µg/mL. Therefore, we further investigated the combined effect of polyhydroxysteroid glycosides at 12.5 µM and sulfated laminaran at 10, 20, and 40 µg/mL.
It was shown that PL1 or PL2 in combination with AaLs at low sub-cytotoxic concentrations did not influence viability or size of 3D HCT 116 spheroids ( Figure 3A,B). On the other hand, the sulfated laminaran AaLs was found to enhance the antiproliferative effect of polyhydroxysteroid glycoside L1 ( Figure 3C). The type of interaction of L1 and AaLs was determined by the Chou-Talalay method for drug combination. The combination index (CI) or fraction affected (Fa) indicated the degree of drug interaction was 0.54, 0.34, and 0.33 or 0.68, 0.59, and 0.52, respectively, which confirmed the synergism of the combined anticancer effect of L1 with AaLs on viability of the 3D HCT 116 spheroids ( Figure 3D). 3D HCT 116 spheroids were treated with PL1 (A), PL2 (B), and L1 (C) (12.5 μM) in combination with AaLs 20, and 40 μg/mL) for 72 h. Cell viability was assessed using the MTS assay. Photos (n = 6 for control or ce treated by PL1, PL2, or L1 with AaLs, where n = the number of photos) of each spheroid were made using ZOE™ Fluorescent Cell Imager and the analysis of spheroids was conducted using ImageJ software bundled 64-bit Java 1.8.0_112 (NIH, Bethesda, Maryland, USA)). (D) Type of combination of L1 with AaLs calculate Compusyn software 1.0.1 (ComboSyn, Inc., Paramus, NJ, USA). Combination index (CI) is a quantitative measure of the degree of interaction between different treatments. A CI equal to 0.9-1.1 is considered additiv CI value of greater than 1.1 represents antagonism; and CI values less than 0.7 denotes synergism. All analy were performed in three independent experiments. Results are expressed as mean ± standard deviation (SD).
Student's t test was used to evaluate data with the following significance level *** p < 0.001.

The Individual and Combined Effects of Sulfated Laminaran and Polyhydroxysteroid Glycosides on Colony Formation of 3D HCT 116 Spheroids
The individual and combined effects of polyhydroxysteroid glycosides PL1, PL2, L1 and sulfated laminaran Aa on the model of growth of the colony of 3D HCT 116 spheroids were determined in soft agar. It was shown tha PL1 at 12.5, 25, and 50 μM by itself inhibited growth of the colony by 4%, 9%, and 24%, respectively, compar the nontreated spheroids (control) (Figure 4A). The individual inhibiting effect of steroidal monoglycoside PL2 the colony growth of 3D HCT 116 spheroids was weak at concentrations up to 50 μM; the percentage of inhibit . Combination index (CI) is a quantitative measure of the degree of interaction between different treatments. A CI equal to 0.9-1.1 is considered additive; a CI value of greater than 1.1 represents antagonism; and CI values less than 0.7 denotes synergism. All analyses were performed in three independent experiments. Results are expressed as mean ± standard deviation (SD). The Student's t test was used to evaluate data with the following significance level *** p < 0.001.

The Individual and Combined Effects of Sulfated Laminaran and Polyhydroxysteroid Glycosides on Colony Formation of 3D HCT 116 Spheroids
The individual and combined effects of polyhydroxysteroid glycosides PL1, PL2, L1 and sulfated laminaran AaLs on the model of growth of the colony of 3D HCT 116 spheroids were determined in soft agar. It was shown that PL1 at 12.5, 25, and 50 µM by itself inhibited growth of the colony by 4%, 9%, and 24%, respectively, compared to the nontreated spheroids (control) ( Figure 4A). The individual inhibiting effect of steroidal monoglycoside PL2 on the colony growth of 3D HCT 116 spheroids was weak at concentrations up to 50 µM; the percentage of inhibition was less than 15% compared to the control ( Figure 4A). L1 at the same doses was found to effectively reduce the size of 3D HCT 116 spheroids by 16%, 38%, and 61%, respectively, compared to the control ( Figure 4A). Sulfated laminaran AaLs at concentrations of 100, 200, and 400 µg/mL was demonstrated to inhibit the growth of the colony of 3D cell cultures by 6%, 20%, and 30%, respectively ( Figure 4B). polyhydroxysteroid glycoside L1 with sulfated laminaran AaLs. It was shown that L1 (12.5 μM) in combination with AaLs at concentrations of 10, 20, and 40 μg/mL possessed a potent anticancer effect, inhibiting the colony growth of 3D spheroids by 25%, 36%, and 80%, respectively, compared with the individual action of L1 ( Figure  4G,H). Taking into account the values of the CI (0.46, 0.37, 0.05) and Fa (0.49, 0.42, and 0.04), the synergism of the combined effect of L1 and AaLs was revealed in the model of colony growth in soft agar (Figure 4J).

The Individual and Combined Effects of Sulfated Laminaran and Polyhydroxysteroid Glycosides on Invasion of 3D HCT 116 Spheroids
There are various invasive assays in the literature, most of which differ in the composition of the polymer matrix that forms an artificial barrier. In this work, a commercial preparation of ECM gel ("Sigma-Aldrich", Saint Louis, MO, USA) was used to create a polymer matrix, which is a standardized mixture of the extracellular matrix proteins obtained from Engelbreth-Holm-Swarm murine sarcoma.
The 3D HCT 116 spheroids were shown to significantly invade into the polymer matrix after 72 h of their incubation ( Figure 5A,F). PL1 by itself at concentrations of 12.5, 25.0, and 50.0 µM decreased the invasion area of spheroids by 20%, 23%, and 37%, respectively, compared to the control ( Figure 5B,F). PL2 did not influence the process of invasion of 3D HCT 116 spheroids up to 50 µM ( Figure 6C,F). The polyhydroxysteroid glycoside L1 at 50 µM possessed the most significant anti-invasive activity and at 50 µM almost completely prevented the invasion of spheroids into the matrix after 72 h of incubation ( Figure 6D,F). It was found that sulfated laminaran AaLs at concentrations of 100, 200, and 400 µg/mL individually inhibited the process of the 3D HCT 116 spheroids' invasion of by 40%, 54%, and 59%, respectively ( Figure 5E,F). Photos of spheroids were made using a Motic microscope (40× magnification, 500 μm sc (F) Quantitative measurement of the invasion area of spheroids treated with individual compounds was carri using the ImageJ program and determined as the difference between total area invaded by cells leaving t spheroid and the area of the spheroid. All analyses were performed in three independent experiments. Resul expressed as mean ± standard deviation (SD). The Student's t test was used to evaluate data with the follow levels of significance *** p < 0.001.  Since linckoside L1 demonstrated the highest individual anti-invasive activity among the investigated polyhydroxysteroid glycosides, it was chosen for the study of the combined effect with a sulfated derivative of laminaran AaLs. It was found that the treatment of 3D HCT 116 spheroids by L1 (12.5 μM) in combination with AaLs (10, 20, and 40 μg/mL) led to the reduction of the invasion area of the spheroids by 16%, 26%, and 44%, respectively, compared to the area of spheroids treated by L1 individually (Figure 6A,B). It was shown that derivative of laminaran AaLs at a low concentration of 10 and 20 μg/mL did not potentiate the effect of L1; the type of action was near additive (Figure 6C). The synergistic anti-invasive effect was observed only when 3D HCT 116 spheroids were treated by L1 at 12.5 μM and AaLs at 40 μg/mL ( Figure 6C).

Mar. Drugs 2021, 19, x FOR PEER REVIEW
Metastasis is a consequence of tumor progression based on the disturbance of survival/apoptosis balance in cancer cells [29]. One of the possible ways of human colorectal carcinoma progression and, consequently, its metastasis is the overexpression and activation of the survival factor serine/threonine kinase, AKT, also known as protein kinase Since linckoside L1 demonstrated the highest individual anti-invasive activity among the investigated polyhydroxysteroid glycosides, it was chosen for the study of the combined effect with a sulfated derivative of laminaran AaLs. It was found that the treatment of 3D HCT 116 spheroids by L1 (12.5 µM) in combination with AaLs (10, 20, and 40 µg/mL) led to the reduction of the invasion area of the spheroids by 16%, 26%, and 44%, respectively, compared to the area of spheroids treated by L1 individually (Figure 6A,B). It was shown that derivative of laminaran AaLs at a low concentration of 10 and 20 µg/mL did not potentiate the effect of L1; the type of action was near additive ( Figure 6C). The synergistic anti-invasive effect was observed only when 3D HCT 116 spheroids were treated by L1 at 12.5 µM and AaLs at 40 µg/mL ( Figure 6C).
Metastasis is a consequence of tumor progression based on the disturbance of survival/apoptosis balance in cancer cells [29]. One of the possible ways of human colorectal carcinoma progression and, consequently, its metastasis is the overexpression and activation of the survival factor serine/threonine kinase, AKT, also known as protein kinase B (PKB). The activation of AKT leads the apoptosis suppression and the uncontrolled proliferation of cancer cells [30][31][32]. AKT is activated by phosphorylation on Thr308 or Ser473 by different stimuli (growth factors, carcinogens, and radiation) and inactivates members of the cell death machinery, such as proteins of the Bcl-2 family [33]. Several members of the Bcl-2 family (including Bcl-2, Bcl-XL, Mcl-1, A1, and Bag-1) promote survival while other members (including Bcl-XS, Bad, Bax, and Bak) promote cell death [34].
It has been proposed that activated AKT phosphorylates Bad at Ser136 block Bad from binding to Bcl-XL or Bcl-2 and promote survival by preventing the concomitant generation of Bax homodimers [35]. So, we supposed that the molecular mechanism of the combined anticancer effect of sulfated derivative of laminaran AaLs from A. angusta and linckoside L1 from P. lincki is associated with the regulation of the activity of AKT kinase, and the major players of apoptosis, namely proapoptotic (Bax), antiapoptotic (Bcl-XL) proteins, and the executioner of apoptosis-caspase 3 and cleaved caspase 3. As shown in the Figure 6D,E, there was a strong activation of AKT kinase in nontreated 3D HCT 116 spheroids (control). Investigated compounds L1 and AaLs alone did not significantly influence the activity of p-AKT kinase (Ser 473) and apoptotic proteins ( Figure 6D,E). On the other hand, L1 (12.5 µM) in combination with AaLs (20 and 40 µg/mL) effectively inhibited phosphorylation of AKT kinase, followed by the up-regulation of the expression level of the proapoptotic protein Bax and the down-regulation of the expression of the antiapoptotic Bcl-XL protein ( Figure  6D,E). This leads to the induction of apoptosis by the activation of an effector caspase 3, and cleaved caspase 3 responsible for the cleavage of the number of death substrates leads to the well-known characteristic hallmarks of an apoptotic cell, including DNA fragmentation, nuclear fragmentation, membrane blebbing, and other morphological and biochemical changes. However, further additional experiments, such as the use of PI3K inhibitor, cell cycle analysis, and DNA comet assay, are needed to confirm the involvement of the PI3K/AKT pathway and the induction of apoptosis in the combined anticancer effect of sulfated laminaran from the brown alga A. angusta and polyhydroxysteroid glycosides from the starfish P. lincki.
In conclusion, we confirmed that the sulfation of laminaran allows increasing its anticancer activity in vitro compared to the activity of native laminaran. Additionally, it was determined that the in vitro anticancer activity of polyhydroxysteroid glycosides PL1 and L1 was more pronounced than the PL2 one in the model of 3D HCT 116 spheroids. PL1 and L1 have similar structural features: 3β,4β-hydroxyl groups in ring A of the steroidal nucleus and the 2-O-methyl-β-D-xylopyranose residue attached to C-3 of the steroid nucleus, while compound PL2 has another ring A of the steroidal nucleus and the α-L-arabinofuranose residue attached to C-24 of the side chain of the steroid aglycone. Probably, indicated above, the structural features of PL1 and L1 contributed to their significant anticancer activity.
The combination of sulfated derivative of laminaran AaLs from the brown alga A. angusta with linckoside L1 from the starfish P. lincki inhibits colorectal cancer progression in vitro, and it is much more effective than the individual effect of these compounds at low, nontoxic concentrations. Moreover, this study, for the first time, provides strong evidence that AaLs in combination with L1 suppressed the proliferation, colony formation, and invasion of 3D HCT 116 spheroids via blocking Akt kinase activity and triggered the apoptosis of colorectal carcinoma cells.
It enriches our understanding of the molecular mechanism of the anticancer action of polysaccharide derivatives of brown algae and polyhydroxysteroid glycosides of starfish, opening up prospects for the development of new approaches for therapy of advanced colorectal cancer in the future. We hope that the obtained data will be of great interest, particularly to researchers working on marine natural compounds and oncology.

Brown Alga
The sample of the alga A. angusta (Aa) was collected from Simushir Island in August 2016, Sea of Okhotsk during a cruise of the research vessel "Academik Oparin". A thallus of fresh algal biomass (100 g) was washed thoroughly with tap water, powdered, and pretreated with 70% aqueous ethanol (w/v = 1:10) for 10 days. Then defatted alga was air-dried at room temperature.

Isolation of Laminaran from A. angusta
The laminaran AaL was isolated from the brown alga A. angusta by the methods as described previously [23].

The Sulfation of Laminaran from A. angusta
Native laminaran AaL from A. angusta was modified by chlorosulfonic acid/pyridine method to obtain the sulfated laminaran AaLs as described earlier [14].

The Structural Characteristics of Sulfated Laminaran
The content of carbohydrates was determined according to the method of Michel Dubois et al. [36]. The absorbance was measured at 490 nm using a Power Wave XS microplate reader (BioTek, Winooski, VT, USA). The glucose (1 mg/mL) was used as a reference standard.
The content of the sulfate group was determined by the turbidimetric method after hydrolysis of the sulfated laminaran AaLs with HCl (1N) [37]. The absorbance was measured at 360 nm using a Power Wave XS microplate reader (BioTek, Winooski, VT, USA). K 2 SO 4 (1 mg/mL) was used as a reference standard.
The 13 C NMR spectra of native and sulfated laminarans were obtained on an Avance DPX-500 NMR spectrometer ("Bruker BioSpin Corporation", Billerica, MA, USA) at 50 • C. The sample concentration was 15 mg of polysaccharide/mL of D 2 O.

Polyhydroxysteroid Glycosides Isolation
Polyhydroxysteroid glycosides PL1, PL2, and L1 were isolated according to the scheme described earlier [21]. In brief, the starfish (500 g) were crushed and extracted twice with methanol (1.0 L) and then twice with ethanol (1.0 L) at room temperature. The extract was concentrated under a vacuum and dissolved in water (2.0 L). Then, the compounds were divided on a polychrome column and chromatographed on a silica gel column in a chloroform-ethanol system (stepwise gradient from 5:1 to 1:5). Nine obtained fractions enriched in polar steroid compounds and their glycosides were subsequently chromatographed on a Florisil column using a chloroform-ethanol system (step gradient from 6:1 to 1:2). The resulting subfractions were further chromatographed using HPLC on reverse phase columns: Diasfer-110-C18 (65% EtOH, 2.5 mL min) and Discovery C18 (65% EtOH, 2.5 mL/min). As a result, the individual compounds were isolated: PL1 (3.5 mg, t R 25.2 min), PL2 (2.5 mg, t R 21.9 min), and L1 (3.0 mg t R 8.9 min). The pbtained PL1, PL2, and L1 were identified based on their thin-layer chromatography (TLC; R f , system BuOH:EtOH:H 2 O 4:1:2), HPLC (t R , Discovery C18 column, 65% EtOH) nuclear magnetic resonance (NMR), and mass spectrometry (MS) data.

Preparation of Compounds for Investigations of In Vitro Anticancer Activity
The vehicle control is the cells treated with the equivalent volume of phosphatebuffered saline (PBS) or dimethyl sulfoxide (DMSO;final concentration was less than 0.5%) for all of the presented experiments.
Polyhydroxysteroid glycosides PL1, PL2, and L1 were dissolved in sterile DMSO to prepare stock concentrations of 40 mM.

Cell Lines and Culture Conditions
The human colorectal carcinoma cells HCT 116 (ATCC ® CCL-247™) were obtained from the American Type Culture Collection (Manassas, VA, USA). HCT 116 cells were cultured in in McCoy's 5A medium supplemented by 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin-streptomycin solution at 37 • C in a humidified atmosphere containing 5% CO 2 . The passage number was carefully controlled and the mycoplasma contamination was monitored on a regular basis.

Formation of Spheroids (3D Cell Culture) by Liquid Overlay Technique (LOT)
The 3D HCT 116 spheroids were formed by the liquid overlay technique (LOT) method with slight modifications. Briefly, to create nonadherent surfaces for the efficient spheroids' formation, 50 µL of preheated (60 • C) agarose (1.5%) was overlaid the bottom of 96-well plates and left to solidify for 1 h at room temperature under sterile conditions.
To study the combined effect of PL1, PL2 or L1 with AaLs, the 3D HCT 116 spheroids were treated by compounds in fixed concentration ratios, which were chosen according to their IC 50. Namely, spheroids were pretreated by PL1, PL2 or L1 at 12.5 µL for 24 h and then AaLs at 10, 20, and 40 µg/mL for an additional 48 h. The metabolic activity of the 3D spheroids after the combined treatment were measured by MTS assay as described above.

Determination of Colony-Inhibiting Activity by Soft Agar Assay
The 3D HCT 116 spheroids were treated by replacing 100 µL of supernatant with a complete medium containing individual compounds: polyhydroxysteroid glycosides PL1, PL2, and L1 at 12.5-50 µM and AaLs at 200-800 µg/mL for 24 h, or their combinations PL1, PL2 or L1 at 12.5 µL for 24 h and then AaLs at 10, 20, and 40 µg/mL for an additional 48 h. The spheroids (n = 6) from each treatment were collected and applied onto 0.3% basal medium eagle (BME) agar containing 10% FBS, 2 mM L-glutamine, and 25 µg/mL gentamicin. The cultures were maintained at 37 • C, in a 5% CO 2 incubator for 21 days, and the cell's colonies were scored using a microscope Motic AE 20 (XiangAn, Xiamen 361101, China) and the ImageJ software.

Determination of Anti-Invasive Activity by 3D Spheroids Invasion Assay
After the 3D HCT 116 spheroids were formed, they were treated by replacing 100 µL of supernatant with a complete medium containing individual compounds: polhxydroxysteroid glycosides PL1, PL2, and L1 at 12.5-50 µM and AaLs at 200-800 µg/mL for 24 h, or their combinations PL1, PL2 or L1 at 12.5 µL for 24 h and then AaLs at 10, 20, and 40 µg/mL for an additional 48 h. The 3D HCT 116 spheroid from each treatment was collected into the 1.5 mL microcentrifuge tubes and left for sedimentation for 10 min. Then, the supernatants were aspirated, 100 µL of cooled ECM gel from an Engelbreth-Holm-Swarm murine sarcoma ("Sigma-Aldrich", Saint Louis, MO, USA) was added to the spheroid and transferred to 96-well plate covered by 100 µL of ECM gel (4 • C). The spheroids were maintained at 37 • C, in a 5% CO 2 incubator for 96 h. A photo of the 3D HCT 116 spheroids (40 × 500 µm scale) was made at the time points of 0, 24, 48, and 72 h with the aid of a microscope Motic AE 20. A quantitative assessment of the invasion of spheroids was carried out using the ImageJ program and was determined as the difference between the total area invaded by the cells leaving the spheroid and the area of the spheroid as described previously [39].

Western Blotting Assay
To determine the effect of the combined treatment with the investigated compounds on the activity of kinases and apoptotic proteins, the 3D HCT 116 spheroids were treated by combinations of PL1, PL2 or L1 at 12.5 µL and AaLs at 10, 20, and 40 µg/mL as described above in Section 3.4.5. "Determination of anti-invasive activity by 3D spheroids invasion assay". The spheroids (n = 9) from each treatment were collected into the 1.5 mL microcentrifuge tubes and left for sedimentation for 10 min. The spheroids were washed by PBS and then lysed by 1X lysis buffer ("Cell Signaling Technology", Danvers, MA, USA) according to the manufacturer's protocol. The Lysate protein was extracted and blotted (20-40 µg) as described previously by Vishchuk et al. [40]. The membranes were treated with the primary antibodies against p-AKT (Ser 473), AKT, Bax, Bcl-XL, caspase 3, cleaved caspase 3, and β-actin ("Cell Signaling Technology", Danvers, MA, USA) and a secondary antibody from a rabbit or a mouse ("Sigma-Aldrich", St. Louis, MO, USA), according to the manufacturer's protocol. Protein bands were visualized using an enhanced chemiluminescence reagent (ECL) ("Bio Rad", Hercules, CA, USA) according to the manufacturer's protocol.

Statistical Analysis
All of the assays were performed in at least three independent experiments. Results are expressed as the mean ±standard deviation (SD). The Student's t test was used to evaluate the data with the following significance levels: * p < 0.05, ** p < 0.01, *** p < 0.001.