## 5 Studies and Mechanism about n-3 PUFAs and Melanoma
### In Vitro Studies
demonstrated that n-3 PUFA regulate COX-2 mRNA expression and PGE2 production in brain metastatic melanoma. Notably, COX-2 mRNA expression decreased in cells incubated with EPA or DHA [73,148]. In contrast, n-6 PUFA increased cell invasion, whereas EPA or DHA did not, highlighting the potential of n-3 PUFA over other PUFAs. These findings demonstrate that incubation with EPA or DHA led to the downregulation of both COX-2 mRNA and protein expression, resulting in a subsequent decrease in invasion in brain-metastatic melanoma [73]. [PERSON] and [PERSON] studied the cytostatic and cytotoxic effects of different non-steroidal anti-inflammatory drugs (NSAIDs) in human skin melanoma A-375 cells, such as celecoxib, indomethacin, aspirin and piroxicam. The study revealed that both celecoxib and indomethacin demonstrated inhibitory properties against cell proliferation, with additive effects observed when cells were co-incubated with DHA. Nonetheless, the anticancer mechanisms of NSAIDs cannot be solely attributed to their COX-inhibitory activities, as only celecoxib and indomethacin exhibited cytostatic or cytotoxic effects on the cancer cells. However, aspirin enhanced the DHA-induced growth inhibition as well as piroxicam [149] (Table 1). DHA increases the expression of the microphthalmia transcription factor (MITF), which prevents \(\beta\)-catenin/TCF from activating metalloproteinase genes such as membrane type 1-MMP, MMP-2 and MMP-13 (Table 1 and Figure 5). Another study reported that DHA not only hindered cell growth and prompted differentiation of melanoma cells in vitro but also demonstrated an anti-neoplastic role associated with the presence of MITF, an oncogene that is involved in melanoma and other malignancies and plays a major role in melanocytic differentiation and survival/proliferation, as well as in oncogenesis [150,151]. This MITF presence interfered with the transcription of pro-invasive target genes. Furthermore, the study revealed that DHA exhibited inhibitory effects on anchorage-independent cell growth, diminished migration and invasion in vitro, and led to the downregulation of various MMPs such as MMP-2, MT1-MMP, MMP-9 and MMP-13, which are known to play a role in melanoma invasion. These MMPs are considered promising molecules for the management of melanoma patients due to their roles as biomarkers and therapeutic targets [150,152]. The role of \(\beta\)-catenin in the anti-cancer effect of n-3 PUFAs is complex and different between the types of cancer. The abnormal Wnt/\(\beta\)-catenin signaling pathway plays a pivotal role in tumor formation and response to therapy by promoting the renewal, proliferation and differentiation of cancer stem cells as well as advancement of malignant progression [153,154]. In the case of melanoma, these effects are related to heightened expression of MITF. DHA also reduces the expression of total \(\beta\)-catenin in metastatic melanoma cell line as well as decreases in cytosolic \(\beta\)-catenin and increases its translocation to the nuclei [148,150]. [PERSON] et at. also showed that DHA and EPA induce apoptosis in melanoma cells through the caspase-3 pathway by increasing Bax proteins and decreasing Bcl-2 proteins, therefore augmenting the Bax/Bcl-2 ratio. In this study, a pro-apoptotic effect was observed for DHA and EPA and not observed with the other fatty oleic acid, suggesting that the pro-apoptotic effect was specific for n-3 PUFAs [148]. These effects on Bcl-2 family proteins have been documented in prior investigations, such as in vitro studies of colorectal cancer [155,156] (Table 1). Oxidative stress plays a pivotal role across all stages of melanoma progression and is intricately involved in both melanin synthesis and the formation of melanoma. Melanoma exhibits heightened susceptibility to oxidative stress, attributed to the interplay of melanin synthesis and UV radiation in the generation of ROS. The impact of oxidative stress extends to melanoma immunity, the metastatic potential of melanoma cells, and their resistance to therapeutic interventions.