**3. Discussion**

Cancer is the leading cause of death worldwide, and colorectal cancer has become a common type of cancer. Given the high incidence, morbidity, and mortality of colorectal cancer, significant research effort is focused on its prevention and treatment. The current work documented that fucoidan affects the migration, apoptosis, and cell cycle progression of colorectal cancer-derived HT-29 cells. Among these effects, the induction of apoptosis was the most potent one. Further analysis of the mechanisms implicated in triggering apoptosis indicated that fucoidan induced this process in HT-29 cells through simultaneous activation of the intrinsic and extrinsic pathways by the JNK signaling pathway.

In the presence of 800 μg/mL of fucoidan, the cell survival rate was approximately 40%, and the induction of apoptosis in HT-29 cells was accompanied by the increase in expression of DR4 at the mRNA and protein levels. After silencing DR4 with siRNA, the difference in the expression of DR4 gradually decreased after inhibiting the extrinsic pathway. Although the expression of the protein was not significantly different from that when DR4 is not silenced, the fraction of surviving cells increased from 40% to 75% at 800 μg/mL of fucoidan, further documenting the critical role DR4 played in the apoptosis of HT-29 cells. In addition, the red-to-green ratio of JC-1 fluorescence decreased from 1.3 to 0.6, suggesting that fucoidan can destroy mitochondrial membrane integrity. These changes triggered the intrinsic apoptotic pathway by the release of cytochrome C into the cytoplasm.

To explore the relationship between the intrinsic and extrinsic pathways, inhibitor of cytochrome C inhibitors and siRNA targeting DR4 siRNA were added. In this experiment, at 800 μg/mL, the cell survival rate was increased from 40% to about 75% when cells were treated only with DR4 siRNA. When the intrinsic pathway or both pathways were inhibited simultaneously, the cell survival rates were improved without significant differences, both from 40% to about 80%, which were higher than the cell survival rates after inhibiting the extrinsic pathway. In addition, the difference in the expression of cytochrome C gradually decreased after inhibiting the extrinsic pathway; the ratio of the

expression of cytochrome C in the experimental group to that of the control group decreased from 1.56 to 1.32 at the concentration of 800 μg/mL. When the intrinsic pathway or both pathways were inhibited simultaneously, the di fference in cytochrome C expression was significantly reduced without significant di fferences, and the ratio decreased from 1.56 to 1.15 and 1.13, respectively, at 800 μg/mL, both lower than the ratio after inhibiting the extrinsic pathway. Therefore, the ratios of cytochrome C expression and cell survival after inhibiting the intrinsic pathway were similar to those after inhibiting both pathways, which were di fferent from those after inhibiting the extrinsic pathway, indicating that the intrinsic pathway was in the downstream of the extrinsic pathway.

It was established that fucoidan inhibited the proliferation activity of HT-29 cells through simultaneous activation of the intrinsic and extrinsic pathways by the JNK signaling pathway, and the intrinsic pathway was downstream of the extrinsic pathway. However, there was still much controversy about whether the intrinsic and extrinsic pathways played an important role in inducing apoptosis in HT-29 cells owing to the lack of untransfected control within the same experiment, which must be improved in order to ge<sup>t</sup> more convincing results. In addition, a study showed that the cell survival rate was only approximately 80% with cells treated with cytochrome C inhibitor or with cytochrome C inhibitor and DR4 siRNA simultaneously; not all cells survived. On one hand, the silencing of DR4 or inhibition of the expression of cytochrome C could not be completed; on the other hand, fucoidan can play other roles besides inducing apoptosis, such as fucoidan significantly blocking the cell cycle, while inducing apoptosis with the increase in concentration. Therefore, future studies should address the impact of fucoidan on both apoptosis and cycle of HT-29 cells. These studies may reveal the inevitable connection between cell cycle and apoptosis and further clarify the mechanism of the induction of HT-29 cell death by fucoidan.
