Pentoxifylline and Norcantharidin Synergistically Suppress Melanoma Growth in Mice: A Multi-Modal In Vivo and In Silico Study

Background: Melanoma is a highly aggressive skin cancer with limited therapeutic re-sponse. Targeting intracellular signaling pathways and promoting tumor cell differen-tiation are promising therapeutic strategies. Pentoxifylline (PTX) and norcantharidin (NCTD) have demonstrated antitumor properties, but their combined mechanisms of action in melanoma remain poorly understood. Methods: The effects of PTX (30 and 60 mg/kg) and NCTD (0.75 and 3 mg/kg), administered alone or in combination, in a DBA/2J murine B16-F1 melanoma model via intraperitoneal and intratumoral (IT) routes were evaluated. Tumor growth was monitored, and molecular analyses included RNA se-quencing, immunofluorescence quantification of PI3K, AKT1, mTOR, ERBB2, BRAF, and MITF protein levels, and molecular docking simulations were performed. Results: Combination therapy significantly reduced tumor volume compared to monotherapies, with relative tumor volume decreasing from 18.1 in the IT control group to 0.6 in the IT combination-treated group. RNA-seq revealed over 3,000 differentially expressed genes in intratumoral treatments, with enrichment in pathways related to oxidative stress, immune response, and translation regulation (KEGG and Reactome analyses). Minimal transcript-level changes were observed for BRAF and PI3K/AKT/mTOR genes; however, immunofluorescence showed reduced total and phosphorylated levels of PI3K, AKT1, mTOR, BRAF, and ERBB2. MITF protein levels and pigmentation increased, especially in PTX-treated groups, indicating enhanced melanocytic differentiation. Docking analyses predicted direct binding of both drugs to PI3K, AKT1, mTOR, and BRAF, with affinities ranging from -5.7 to -7.4 kcal/mol. Conclusions: The combination of PTX and NCTD suppresses melanoma progression through dual mechanisms: inhibition of PI3K/AKT/mTOR signaling and promotion of tumor cell differentiation.