Synergistic Antiproliferative Effects of Chondroitin Sulfate and Fucoidan in Tumor-derived Spheroids: Insights From a 3D Cell Culture Approach

In our experiments the spheroids were prepared using Liquid overlay technique (Nováková et al., 2023). Following the formation of spheroids from NIH3T3 (Fig. 1B) and Hepa1c1c7 (Fig. 2B) cell lines on the third day of culture, treatments were initiated using the tested substances CS, fucoidan (F), and their combination (CS + F). These compounds were diluted in DMEM culture medium at ratios of 1:1, 1:2, and 1:4, and the specific concentrations of each dilution are detailed in Table 1. Control cells were treated with PBS. At intervals 3rd, 5th and 7th day of the cultivation, 100 μL of medium was aspirated from each well of a 96-well plate and an equivalent volume of the respective treatment was added. Before each addition, the spheroids were observed under an inverted microscope (Zeiss Axiovert 40 CFL, with AxioCam ICc3 camera, Zeiss, Germany) at a magnification of 50×. Images were captured using the ProView software, and the area of the spheroids was measured in pixels (px) utilizing ImageJ software (Schneider et al., 2012). The experimental duration was 10 days (after which the spheroids began to disintegrate) during which substances CS, fucoidan, or their combination (CS + F) were administered to the spheroids three times at 3rd, 5th and 7th day of the cultivation. The results presented are averages derived from three independent experiments.

Figure 2.

Comparison of the Hepa1c1c7 tumor cell line in 2D and 3D cell culture: A) Hepa1c1c7 tumor cell line in 2D cell culture forming a cell monolayer (100× magnification); B) Hepa1c1c7 tumor cell line in 3D cell culture forming a spheroid (100× magnification).

Figure 3.

Spheroids from the non-tumor cell line NIH3T3 after treatment with A) chondroitin sulfate (CS), B) fucoidan (F), C) a combination of chondroitin sulfate and fucoidan (CS + F). Day 3 – first addition of substances; Day 7 – last addition of substances. Spheroids were photographed at 50× magnification. Up arrow (↑) – spheroids that increased their area compared to control spheroids; down arrow (↓) – spheroids that decreased their area compared to control spheroids; equal to (=) – spheroids that have approximately the same area compared to control spheroids.

The flow cytometry method was employed to quantitatively assess the various cell forms present within the spheroids. Initially, the spheroids, along with the culture medium, were transferred to a 96-well plate with a rounded bottom. Subsequently, the spheroids were washed with PBS and treated with a trypsin-EDTA solution to disrupt the spheroid structure. Following this treatment, the cells were resuspended and fixed. For cell staining, a solution containing propidium iodide was utilized to differentiate between viable and non-viable cells, while annexin V was employed to identify apoptotic cells. Measurement was conducted using the Guava^® EasyCyte™ Plus flow cytometry system (New Life Scientific, OH, USA).

Spheroids generated from the non-tumor cell line NIH3T3 were treated with compounds fucoidan, CS, and their combination (CS + F), while control spheroids were treated with PBS. An increase in the surface area of the spheroids was observed compared to the control cells (Fig. 3). The most significant growth of spheroids was recorded when a combination of both active substances was applied (CS + F), that is, at a concentration of CS 0.0155 mmol/L and fucoidan 0.023 mmol/L (1:4). In contrast, a decrease in the area of the spheroids was noted when treated with fucoidan at a concentration of 0.092 mmol/L (1:1).

Spheroids formed from the Hepa1c1c7 tumor cell line were treated with compounds CS, fucoidan, or their combination (CS + F), while control spheroids were treated with PBS. As depicted in Fig. 4, a gradual increase in size of the spheroid area was observed following treatment with the substances throughout the duration of the experiment. An exception was noted for the spheroids treated with substance fucoidan at a concentration of 0.092 mmol/L (1:1), as their area exhibited only slight changes compared to the control. The greatest growth of spheroids was recorded for the combination of substances (CS + F) at concentrations of 0.0155 mmol/L of CS and 0.023 mmol/L of fucoidan at dilution with DMEM in the ratio 1:4.

Figure 4.

Spheroids from the Hepa1c1c7 tumor cell line after treatment with A) chondroitin sulfate (CS), B) fucoidan (F), and C) a combination of chondroitin sulfate and fucoidan (CS + F). Day 3 – first addition of substances; Day 7 – last addition of substances. Spheroids were photographed at 50× magnification. Up arrow (↑) – spheroids that increased their area compared to control spheroids; down arrow (↓) – spheroids that decreased their area compared to control spheroids; equal to (=) – spheroids that have approximately the same area compared to control spheroids.

To monitor the quantitative representation of live, apoptotic, necrotic, and dead cells in spheroids following treatment with CS, fucoidan, or their combination (CS + F), spheroids were prepared from the non-tumor cell line NIH3T3 and the tumor cell line Hepa1c1c7 for analysis by flow cytometry. Spheroids treated with substances at concentrations of 0.0155 mmol/L (CS) and 0.023 mmol/L (fucoidan), as well as their combination (CS + F), were selected based on results from previous experiments indicating that these concentrations had the most significant effect on spheroid growth. In the case of the non-tumor cell line NIH3T3 (Table 2, Fig. 5), treatment with CS resulted in an increase in the number of live cells (approximately 34.86%) and dead cells (approximately 5.18%), while a decrease in apoptotic cells was observed (approximately 11.69%). Conversely, treatment with substance fucoidan led to a reduction in live cells (approximately 18.04%) and dead cells (approximately 3.11%), but an increase in apoptotic cells was noted (approximately 6.05%). After treatment with a combination of substances (CS + F), a decrease in both live cells (approximately 3.27%) and apoptotic cells (approximately 13.57%) was recorded compared to control cells. In the case of the tumor cell line Hepa 1c1c7 (Table 2, Fig. 6), treatment with CS resulted in a decrease in the number of live cells (approximately 20.82%), apoptotic cells (approximately 3.65%), and dead cells (approximately 3.2%), while an increase in necrotic cells was observed (approximately 27.67%). Conversely, treatment with fucoidan led to a reduction in live cells (approximately 0.35%) and apoptotic cells (approximately 5.56%), but an increase in dead cells (approximately 2.84%) and necrotic cells (approximately 3.06%) was noted. After treatment with a combination of substances (CS + F), a decrease in live cells (approximately 8.48%), apoptotic cells (approximately 1.72%), and necrotic cells (approximately 2.55%) was recorded, compared to control cells. The individual measured values are presented in Table 2. From the results of these experiments, it can be concluded that the most favorable outcomes were observed with the synergy of CS + F at concentrations of 0.0155 mmol/L of CS and 0.023 mmol/L of fucoidan.

Figure 5.

Flow cytometric analysis of non-tumor NIH3T3 cell lines treated with A) chondroitin sulfate (CS), B) fucoidan (F), and C) a combination of chondroitin sulfate and fucoidan (CS + F), and D) phosphate-buffered saline (PBS). Dead cells are shown on the top left, necrotic cells on the top right, live cells on the bottom left, and apoptotic cells are shown on the bottom right.

Figure 6.

Flow cytometric analysis of Hepa1c1c7 tumor cell line treated with A) chondroitin sulfate (CS), B) fucoidan (F), and C) a combination of chondroitin sulfate and fucoidan (CS + F), and D) phosphate-buffered saline (PBS). Dead cells are shown on the top left, necrotic cells on the top right, live cells on the bottom left, and apoptotic cells are shown on the bottom right.

The 3D cell culture was used to monitor the quantitative representation of live, apoptotic, necrotic, and dead cells in spheroids after treatment with CS, fucoidan, and their combination (CS + F). Spheroids from the NIH3T3 cell line formed round spheroids with a hypoxic core with an average area of 945 px and spheroids from the Hepa1c1c7 cell line formed larger spheroids with an average area of 1521 px; the hypoxic core increased in size during the experiment. From the flow cytometry analyses, the highest percentage of live cells in spheroids was recorded after treatment with CS (66.43 ± 4.43%) in the case of the NIH3T3 cell line and in the case of the Hepa1c1c7 tumor cell line treated with fucoidan (49.65 ± 4.77%). On the contrary, in spheroids derived from both cell lines, flow cytometry revealed the highest number of dead cells after treatment with a combination of CS and fucoidan (CS + F) in the case of NIH3T3 (24.23 ± 5.28%) and Hepa1c1c7 (19.84 ± 5.80%). From experimental studies of substances based on GAGs and sulfated heteropolysaccharides, it can be concluded that the synergistic effect of CS and fucoidan on spheroids showed the best results.

As we can notice, there is a contrasting effect observed in non-tumor (NIH3T3) and tumor (Hepa1c1c7) cell lines, particularly the proliferative effect of CS and fucoidan on NIH3T3 cells versus the antiproliferative effects on Hepa1c1c7 cells. The contrasting effects of CS and fucoidan on non-tumor (NIH3T3) and tumor (Hepa1c1c7) cell lines can be attributed to several biological mechanisms and cellular contexts. Non-tumor cells, like NIH3T3, often exhibit enhanced proliferation in response to certain growth factors and polysaccharides, including CS and fucoidan. These compounds can stimulate signaling pathways that promote cell growth and differentiation in normal cells. For instance, fucoidan has been shown to activate pathways such as extracellular signal-related kinase and c-Jun N-terminal kinase, which are involved in the proliferation and differentiation of various cell types, for example, osteoblasts (Kim et al., 2015). In contrast, tumor cells like Hepa1c1c7 may respond differently due to their altered signaling pathways and microenvironment. Tumor cells often have dysregulated growth signals and may also be more sensitive to the inhibitory effects of certain compounds, leading to antiproliferative outcomes when treated with CS or fucoidan (Bittkau et al., 2019; Kiselevskiy et al., 2022). The molecular weight and specific composition of fucoidan can significantly influence its biological activity. Higher molecular weight fucoidans have been associated with enhanced immune responses and may exhibit different effects on tumor versus non-tumor cells. For example, some studies suggest that high-molecular weight fucoidan can activate immune cells, which might contribute to its antiproliferative effects on tumor cells while promoting proliferation in normal cells (Anisimova et al., 2017; Kiselevskiy et al., 2022). The tumor microenvironment itself plays a crucial role in how cancer cells respond to various treatments. Tumor cells often exist in a hypoxic environment with altered nutrient availability, which can affect their metabolism and response to external stimuli like CS and fucoidan. In this context, antiproliferative effects could arise from the inability of tumor cells to adapt to these polysaccharides as effectively as non-tumor cells do (Hayakawa and Nagamine, 2009). Finally, fucoidan has been noted to exhibit cytotoxic properties against certain cancer cell lines by interfering with metabolic processes or inducing apoptosis. This cytotoxicity may not be present in non-tumor cells like NIH3T3, which could explain why these cells proliferate in response to CS and fucoidan while Hepa1c1c7 cells do not (Bittkau et al., 2019; Hayakawa and Nagamine, 2009).

Fucoidans and their derivatives exhibit a wide range of immunomodulatory effects, participating in immune responses against various cancers and infectious diseases. The sulfate and acetyl groups primarily contribute to their activity (Saliba et al., 2023). Our results are consistent with the findings of Liu et al. (2020) who reported that fucoidan has no effect on normal ovarian cells, but inhibits the proliferation of ovarian cancer cells (Liu et al., 2020). Many other studies have confirmed the anticancer effects of fucoidan, highlighting its potential as a therapeutic agent in cancer treatment (Bai et al., 2020; Chantree et al., 2021; Duan et al., 2020; Liu et al., 2020). The principle of the anticancer action of fucoidan lies in its multifaceted mechanisms that target various cellular processes associated with cancer progression. Fucoidan has been shown to promote programmed cell death in cancer cells by activating intrinsic and extrinsic apoptotic pathways. This includes the upregulation of proapoptotic proteins and the activation of caspases, leading to increased apoptosis in various cancer cell lines (Duan et al., 2020). Chantree et al. (2021) reported that fucoidan can inhibit the proliferation of cancer cells by inducing cell cycle arrest. Specifically, it has been reported to cause G0/G1 phase arrest, which prevents cells from progressing through the cell cycle and dividing (Chantree et al., 2021). Finally, fucoidan has anti-inflammatory properties by inhibiting proinflammatory cytokines and enzymes, thereby creating a less favorable environment for tumor growth (Sanjeewa et al., 2021). These mechanisms collectively highlight fucoidan’s potential as a therapeutic agent in cancer treatment, warranting further investigation into its clinical applications.

The application of CS in the field is controversial. Several in vitro studies confirm its proliferative effect on tumor cells. However, some modified forms of CS are being explored for their potential anticancer properties. In this case, further research is necessary to clarify these effects (Asimakopoulou et al., 2008). Our study explored the effects of CS and fucoidan on spheroids derived from tumor cell lines, demonstrating enhanced growth inhibition caused by fucoidan (Fig. 4). The synergistic effect of the combined treatment of CS and fucoidan was confirmed through flow cytometry. The percentage of dead tumor cells increased from 7.09% in the control group (PBS) to 19.84% when both substances CS+F were used together (Table 2). While the use of each substance separately also resulted in a higher percentage of dead cells compared to the control group (PBS), the numbers were not nearly as high as when the two agents were combined.