## 5 Studies and Mechanism about n-3 PUFAs and Melanoma
### In Vivo Studies
In this study, the administration of EPA resulted in elevated phosphorylation levels of JNK and p38, while no observable phosphorylation of ERK was detected, in accordance with studies described above. In the presence of 5-FU treatment, the cell viability in EPA-treated cells exhibited a significant decrease compared to untreated cells. However, when the expression of Cx43 was suppressed, the combination therapy did not induce additional cytotoxic activity. These findings imply that Cx43 plays a crucial role in the susceptibility of cells to 5-FU (Figure 4). Likewise, the combined treatment of EPA and 5-FU effectively suppressed tumor growth, prolonged the survival of mice with tumors, and resulted in an elevated number of apoptotic cells in the EPA-treated tumors, as observed in sections from B16-F10-bearing mice [129] (Table 1). Different research demonstrates that n-3 PUFA can act as agonists of GPR, namely, GPR40/free fatty acid receptor (FFA) 1 and GPR120/FFA4, which is a promising target in several pathologies such as obesity, type 2 diabetes, inflammation-related diseases and cardiovascular diseases, among others [18; 169; 168]. Particularly in melanoma, [PERSON] et al. investigated whether the G protein-coupled receptors such as GPR40, which is expressed at high levels in several human melanoma cell lines, are a potential therapeutic target in melanoma. They used human melanoma and control fibroblast cells for in vitro studies, which were treated with DHA and TAK-875 and GW9508 (selective CPR40 agonist and non-selective GPR agonist that stimulate GPR120 and GPR40, respectively). A murine subcutaneous xenograft model of human melanoma was employed to assess the impact of dietary intervention with an n-3 PUFA-enriched diet in comparison to an n-6 PUFA-enriched diet on the progression of human melanoma in vivo. Additionally, a comparable animal model was utilized to evaluate the efficacy of oral TAK-875 in inhibiting the growth of established melanoma tumors in vivo. Their findings revealed that DHA exhibits inhibitory effects on the proliferation of human melanoma cells both in vitro and in vivo. Tumors derived from subjects receiving the n-3 PUFA-rich diet exhibited a reduction in weight by 69% and in volume by 76% compared to those from subjects on the n-6 PUFA-rich diet. Furthermore, TAK-875 demonstrated inhibitory effects on human melanoma cell growth both in vitro and in vivo. Specifically, tumors from subjects treated with TAK-875 displayed reductions in weight by 46% and in volume by 62% and exhibited a 77% slower growth rate compared to the control group [170] (Table 1). Another investigation revealed contrasting effects of fatty acids on melanogenesis in melanoma cells, indicating that saturated fatty acids augment melanogenesis, whereas PUFAs exert a suppressive influence. Specifically, EPA not only diminishes tumor growth but also mitigates F-actin levels by attenuating the phosphorylation of cofilin (Figure 5). This stands in stark contrast to the action of palmitic acid, which induces the formation of F-actin stress fibers through the activation of the RhoA protein [171] (Table 1). This study elucidated a direct interaction between DHA and Receptor for Activated C Kinase 1 (Rack1), prompting the activation of PKC and subsequent initiation of oncogenic signaling pathways (Figure 4). Rack1 is a conserved eukaryotic protein renowned for its multifaceted biological roles in humans, including the facilitation of cyclin binding activities. Notably, the upregulation of Rack1 has been associated with lymph node metastasis in cervical cancer, while PKC-mediated signaling pathways have been implicated across a spectrum of cancer types [172]. In melanoma cells cultured with DHA and PMA (utilized to activate PKC signaling), a decline in the antitumor effect of DHA was observed. Moreover, it is well established that PKC has been documented to upregulate the expression of glucose transporters such as GLUT-1 and GLUT-4 [173]. Indeed, [PERSON] et. al.