## 5 Studies and Mechanism about n-3 PUFAs and Melanoma
### In Vivo Studies
There is also dysregulation of the chemokine receptor genes that are associated with melanoma progression such as CXCR4, which is expressed in a variety of immune cells and cancer tissues, producing ulceration, tumor thickness and lymph node metastasis and enhancing the progression of melanoma [164]. [PERSON] et al. conducted an in vivo experiment with Fat-1 transgenic mice and showed an inverse correlation between lung tissue n-3 PUFA levels with the frequency of melanoma pulmonary metastasis. The Fat-1 group had fewer pulmonary tumor foci and smaller metastatic foci volume in both early and late stages of metastasis and the lung tissue levels of n-3 PUFAs, including EPA, DPA and DHA, were higher in Fat-1 mice than in WT mice. Moreover, pulmonary myeloperoxidase activity was higher in WT mice than in Fat-1 mice and serum levels of immune cell-expressed inflammatory factors with roles in tumor metastasis, such as IL-17 and TNF-\(\alpha\), were lower in Fat-1 mice than in WT mice as well as metastasis-related factors, including CXCR4 and transforming growth factor-\(\beta\), suggesting that n-3 PUFAs that inhibit melanoma pulmonary metastasis involve suppression of pulmonary and systemic inflammation. It can be highlighted that n-6 and n-3 PUFAs exhibit differential effects on CXCR4 expression. They showed that levels of EPA and its derivatives were significantly higher in the serum of Fat-1 mice than in WT mice with a lower ratio of n-5/n-3 PUFAs in Fat-1 mice compared with WT mice and the cells either with EPA or 18-HEPE suppress CXCR4 expression although AA elicits the opposite. In synthesis, n-3 PUFAs can decrease lung metastasis of melanoma by increasing the production of 18-HEPE, which can inhibit CXCR4 expression and inflammatory response level [89] (Table 1 and Figure 5). In vivo studies employing non-Fat-1 transgenic mice have also been conducted, yielding outcomes comparable to those previously described. [PERSON] et al. showed that mice subjected to algal oil treatment, particularly rich in DHA, exhibited suppressed pulmonary metastases and inhibited outgrowth of melanoma cells. This effect was associated with the induction of autophagy, as evidenced by an elevation in LC3-II levels (Figure 3). The mechanism involved the modulation of mammalian target of rapamycin (mTOR) and p38 mitogen-activated protein kinase (MAPK), along with the activation of JNKs, resulting in reduced p62 accumulation and diminished secretion of the proinflammatory cytokine IL-18 [65] (Table 1), which are also related with oxidative stress pathways [165]. These outcomes suggest that the antitumorigenic activities of algal oil are mediated through autophagy-driven elimination of p62 and possess anti-inflammatory properties [65] (Figure 4). Studies demonstrate that in tumor cells there are reduced expressions of Cx43 which are important for enhancing chemosensitivity that is suppressed in a tumor microenvironment [129; 166; 167]. [PERSON] et al. carried out a study where B16-F10 murine melanoma cells were used. These cells were pretreated with various inhibitors for 1 h and then EPA was added. EPA-treated, non-treated or transfected cells were exposed to 5-FU. Moreover, they used mice that were inoculated with B16-F10 cells. Groups of tumor-bearing mice were orally administrated with EPA followed by 5-FU treatment, or with either treatment alone. Exposing cells to escalating concentrations of EPA led to a dose-dependent rise in Cx43 levels compared to controls. The observed correlation between the expression of Cx43 induced by EPA in melanoma cells and the degrees of gap junction intercellular communication implies that EPA may trigger Cx43 expression, potentially enhancing Cx43 functionality in intercellular communication through gap junctions (Figure 5). Moreover, the potential molecular mechanisms in EPA-induced Cx43 expression through the MAPK signaling pathways are described.