RIPK4 knockout enhances the antitumor effects of melatonin pretreatment in melanoma cells in vitro and in a xenograft model in zebrafish

Melanoma is an aggressive skin cancer characterized by high metastatic potential and resistance to conventional therapies. Melatonin, a neurohormone known for its circadian and antioxidant functions, has demonstrated anti-proliferative and pro-apoptotic effects in melanoma through both receptor-dependent and receptor-independent mechanisms. Receptor-interacting protein kinase 4 (RIPK4), a regulator of keratinocyte differentiation and the NF-κB and Wnt/β-catenin signaling pathways, has recently emerged as a contributor to melanoma progression and therapy resistance. In this study, we investigated the interaction between melatonin signaling and RIPK4 expression in human melanoma models. We demonstrate that pharmacological concentrations of melatonin significantly reduce cell viability and proliferation in vitro, with a more pronounced effect in RIPK4-knockout (RIPK4.KO) cells. Interestingly, loss of RIPK4 led to the upregulation of melatonin receptors MT1 and MT2, although pharmacological blockade of these receptors failed to reverse melatonin-induced cytotoxicity, suggesting a predominantly receptor-independent mechanism of action. In the zebrafish xenograft model, melanoma cells pretreated with melatonin prior to injection exhibited reduced tumor growth, and the combination with RIPK4 knockout produced an additive anti-tumor effect. Our findings support a novel functional link between RIPK4 and melatonin sensitivity and highlight the potential of combining RIPK4-targeted strategies with melatonin in melanoma therapy.