A novel liphagal analog, IIIM-321, induces apoptosis in melanoma cells via autophagy modulation and PI3K/MAPK pathway inhibition.

Melanoma is an aggressive and highly metastatic skin cancer characterized by high mortality and resistance to conventional chemotherapy. Aberrant activation of the PI3K and MAPK signaling pathways in melanoma cells enhances survival and drives tumor progression. Targeting autophagy has emerged as a novel therapeutic strategy to induce cell death in melanoma. In this study, we evaluated IIIM-321, a synthetically derived lipidated analog of liphagal, which effectively downregulated both MAPK and PI3K signaling pathways, leading to a time- and dose-dependent reduction in melanoma cell proliferation. IIIM-321 induced significant pro-apoptotic effects in B16F10 cells, accompanied by autophagy inhibition, reactive oxygen species (ROS) accumulation, modulation of antioxidant defenses, and mitochondrial membrane potential (ΔΨM) disruption. The compound induced G0/G1 cell cycle arrest through upregulation of p21, downregulation of Cyclin D1, and its degradation via phosphorylated GSK3β, ultimately triggering apoptosis. Apoptotic induction was further confirmed by annexin-V/PI staining, cytochrome c release, caspase activation, and PARP cleavage. IIIM-321-mediated autophagy inhibition was evidenced by increased LC3-II, decreased Beclin1, and p62/SQSTM1 accumulation. These findings were supported by acridine orange staining and immunocytochemistry for Beclin1 and p62. Additionally, IIIM-321 suppressed both basal and α-MSH-stimulated melanogenesis in B16F10 cells, along with reduced expression of Tyrosinase, Trp-1, and Trp-2. Collectively, our findings establish IIIM-321 as a promising anti-melanoma agent with therapeutic potential for future clinical development. Graphical abstract: Supplementary Information: The online version contains supplementary material available at 10.1007/s10616-025-00834-3.