*2.1. Cytotoxicity of Fucoidan*

Fucoidan administered at concentrations up to 800 μg/mL was not cytotoxic to human normal cell 293T cells (Figure 2A). However, fucoidan induced death of HeLa, MCF-7, and HT-29 cells in a dose-dependent manner (Figure 2B–D). The cytotoxic effect of fucoidan was most pronounced in HT-29 cells, with the cell survival rate of only approximately 40% at 800 μg/mL of the compound. Therefore, HT-29 cells and fucoidan concentrations of up to 800 μg/mL were selected for further experiments.

**Figure 2.** Cytotoxicity of fucoidan. (**A**) Toxicity of fucoidan to 293T cell is expressed as the means ± SD (n = 3). (**B**) Toxicity of fucoidan to HT-29 cells is expressed as the means ± SD (n = 3). (**C**) Toxicity of fucoidan to MCF-7 cell is expressed as the means ± SD (n = 3). (**D**) Toxicity of fucoidan to HeLa cell is expressed as the means ± SD (n = 3). \*, *p* < 0.05; \*\*, *p* < 0.01; \*\*\*, *p* < 0.001.

#### *2.2. Pharmacological Activity of Fucoidan on HT-29 Cells*

To explore the pharmacological effects of fucoidan on HT-29 cells, apoptosis, migration, and cell cycle were analyzed. We can find that the treatment increased the rate of apoptosis of HT-29 cells in a dose-dependent fashion, with 80% of the cells in the late stage of apoptosis at 800 μg/mL of fucoidan (Figure 3A,D). However, fucoidan blocked the cells in the G0/G1 phase of the cell cycle, with 50% of the cells in the G0/G1 phase of the cell cycle at 800 μg/mL of fucoidan, and the fraction of arrested cells increased with higher fucoidan concentrations (Figure 3B,E). Additionally, the migration of HT-29 cells tended to decrease with increasing fucoidan concentration and incubation time, but the reduction in migratory activity did not reach statistical significance, remaining at approximately 30% at 800 μg/mL (Figure 3C,F). These findings indicated that fucoidan affected apoptosis more significantly than migration and cell cycle.

**Figure 3.** Pharmacological activity of fucoidan on cells. (**A**) Detection of apoptosis by flow cytometry. (**B**) Detection of cell cycle by flow cytometry. (**C**) Detection of cell migration. (**D**) Statistical results of apoptosis are expressed as the means ± SD (n = 3). (**E**) Statistical results of cell cycle are expressed as the means ± SD (n = 3). (**F**) Statistical results of cell migration are expressed as the means ± SD (n = 3). \*, *p* < 0.05; \*\*, *p* < 0.01; \*\*\*, *p* < 0.001.

#### *2.3. Analysis of Fucoidan-Induced Apoptosis of HT-29 Cells*

#### 2.3.1. Fucoidan Can Induce Apoptosis Through the Extrinsic Pathway

To explore the involvement of receptors in the activation of apoptosis by fucoidan, the expression of DR4 and related proteins at the transcriptional and translational level was determined. All examined proteins, including DR4 and caspase-3, -6, and -9, were upregulated by fucoidan in a concentration-dependent manner (Figure 4A). The expression level of DR4 increased with the increase of fucoidan concentration at the gene level and the result demonstrated that DR4 was required for the induction of apoptosis by fucoidan (Figure 4B). To determine whether DR4 was required for the induction of apoptosis by fucoidan, siRNA was used to silence its expression, whose silence rate was about 65% (Figure 4C). However, although the expression of all examined proteins was suppressed in the presence of siRNA targeting DR4 (Figure 4D), these proteins did not decrease significantly with the increasing concentration in comparison, which may be because of DR4's low silence rate. However, DR4 silencing decreased the cytotoxicity of fucoidan (800 μg/mL) on HT-29 cells, resulting in an increase in the survival rate from 40% to 75% (Figure 4E). These results demonstrated that fucoidan can induce apoptosis of HT-29 cells by upregulating DR4.

#### 2.3.2. Fucoidan Can Induce Apoptosis Through the Intrinsic Pathway

To determine whether the mitochondrial pathway can contribute to fucoidan-induced apoptosis of HT-29 cells, the changes in mitochondrial membrane potential were determined by the JC-1 probe, and the expression of cytochrome C protein was assessed by Western blotting. It can be seen that, with an increase in fucoidan concentration, the red-to-green ratio of JC-1 fluorescence decreased from 1.3 to 0.6, indicating a reduction in the inner mitochondrial membrane potential and an increase in its permeability (Figure 5A,B). Concurrently, cytochrome C was released from the mitochondria into the cytoplasm; this e ffect was also dependent on fucoidan concentration, and the ratio of cytochrome C expression in the experimental group to the control group was 1.56 at 800 μg/mL (Figure 5C). The release of cytochrome C initiated the caspase cascade, leading to apoptosis. These results showed that fucoidan activated not only the extrinsic pathway through surface death receptors, but also the intrinsic mitochondrial pathway-mediated apoptosis of HT-29 cells.

#### 2.3.3. Relationship Between the Extrinsic and Intrinsic Pathways

Cells in this experiment were divided into three groups: the first group, cells treated with siRNA for DR4; the second group, cells treated with cytochrome C inhibitor; and the third group, cells treated simultaneously with cytochrome C inhibitor and DR4 siRNA. We can find that in cells treated only with DR4 siRNA at 800 μg/mL, the ratio of cytochrome C expression in the experimental group to the control group decreased from 1.56 to 1.32 (Figures 5C and 6A,B), and the cell survival rate was increased from 40% to about 75%( Figures 2B and 6C). When cells were treated only with the inhibitor of cytochrome C or treated simultaneously with cytochrome C inhibitor and DR4 siRNA, there was no significant di fference in the ratio and cell survival rate between the second and third group. In the second and third group, it can be seen that the ratio decreased from 1.56 to 1.15 and 1.13 at 800 μg/mL, respectively (Figures 5C and 6D–H), both lower than the ratio after inhibiting the death receptor pathway; besides, at 800 μg/mL, the cell survival rate was increased from 40% to about 80% ( Figures 2B and 6F,I), both higher than 75%.Therefore, the di fference of cell survival rate and cytochrome C expression indicated that the mitochondrial pathway was downstream of the DR4 pathway.

**Figure 4.** Fucoidan induced apoptosis through DR4. (**A**) Results of Western blotting of proteins. (**B**) Results of Reverse Transcription-Polymerase Chain Reaction (RT-PCR) with DR4. (**C**) Results of Western blotting of proteins with the silent DR4. (**D**) Expression of proteins after DR4 was silenced. (**E**) The toxicity of fucoidan to HT-29 cells with silent DR4 is expressed as the means ± SD (n = 3). \*, *p* < 0.05; \*\*, *p* < 0.01; \*\*\*, *p* < 0.001.

**Figure 5.** Changes of membrane potential induced by fucoidan in HT-29 cells. (**A**) Results of JC-1 staining. (**B**) Results of red-green fluorescence ratio. (**C**) Results of Western blotting of cytochrome C. \*\*, *p* < 0.01; \*\*\*, *p* < 0.001.

**Figure 6.** Effects of cytochrome C inhibitor on HT-29 cells. (**A**) Results of Western blotting of cytochrome C with the silent DR4. (**B**) Results of Western blotting of cytochrome C with cytochrome C inhibitor. (**C**) Results of Western blotting of cytochrome C with cytochrome C inhibitor and silent DR4. (**D**) Results of protein expression ratio at different concentrations with the silent DR4. (**E**) Results of protein expression ratio at different concentrations with cytochrome C inhibitor. (**F**) Results of protein expression ratio at different concentrations with cytochrome C inhibitor and silent DR4. (**G**) The toxicity of fucoidan to HT-29 cells with the silent DR4 is expressed as the means ± SD (n = 3). (**H**) The toxicity of fucoidan to HT-29 cells with cytochrome C inhibitor is expressed as the means ± SD (n = 3). (**I**) The toxicity of fucoidan to HT-29 cells with cytochrome C inhibitor and silent DR4 is expressed as the means ± SD (n = 3). \*, *p* < 0.05; \*\*, *p* < 0.01.

#### *2.4. E*ff*ect of Fucoidan on the JNK Signaling Pathway in HT-29 Cells*

The role of the JNK signal pathway in the induction of apoptosis in HT-29 cells was determined by RT-PCR and Western blotting. At the mRNA level, the expression of ras, raf, MEK1, MEK2, and JNK were upregulated with increasing concentrations of fucoidan (Figure 7A,B). Moreover, fucoidan increased the level of JNK protein and its phosphorylated form, P-JNK, in a dose-dependent fashion, as assessed by Western blot analysis (Figure 7C); the ratio of p-JNK/JNK increased from 2.9 to about 5.2 at 800 μg/mL (Figure 7D). Therefore, the JNK signaling pathway was essential for the activation of apoptosis of HT-29 cells by fucoidan.

**Figure 7.** Effects of fucoidan on JNK signaling pathway in HT-29 cells. (**A**) Results of RT-PCR. (**B**) Ratio of related expression factors at mRNA level. (**C**) Results of Western blotting of related proteins. (**D**) Ratio of JNK to p-JNK. \*, *p* < 0.05; \*\*, *p* < 0.01; \*\*\*, *p* < 0.001.
