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. Inbred DBA/2J mice (6–8 weeks old) were obtained. The animals were housed in polycarbonate cages with sawdust bedding under controlled conditions (23°C, 40–60% humidity) and maintained on a 12-hour light:12-hour dark cycle. Food and water were provided ad libitum, with rodents receiving NUTRICUBOS Rodent Chow Cargill® (SAGARPA Authorization A-0207-246) and potable water. The study included two main experimental groups: (1) an intraperitoneal (IP) treatment group, in which 7 × 10⁵ cells (melanoma B16-F1 cells (ATCC® CRL-6323™) ) were subcutaneously implanted on the right flank of the mouse, and therapeutic agents were administered intraperitoneally; (2) an intratumoral (IT) treatment group, in which 7 × 10⁵ cells ( melanoma B16-F1 cells (ATCC® CRL-6323™) ) were subcutaneously implanted on the right flank of the mouse, and drugs were administered directly into the tumor. On Day 0, cells were implanted, and once a visible and palpable tumor developed, treatment groups were sex-matched and randomly assigned, and pharmacological treatment was initiated. For the IP treatment group, animals were randomly assigned to the following subgroups: Subgroup 1 (control), receiving sterile saline solution; Subgroup 2, receiving PTX at 60 mg/kg; Subgroup 3, PTX at 30 mg/kg; Subgroup 4, NCTD at 3 mg/kg; Subgroup 5, a combination of PTX at 60 mg/kg and NCTD at 3 mg/kg; Subgroup 6, PTX at 60 mg/kg combined with NCTD at 0.75 mg/kg; and Subgroup 7, NCTD at 0.75 mg/kg. For the IT treatment group, animals were divided into four subgroups as follows: Subgroup 1 (control), receiving DMSO; Subgroup 2, PTX at 60 mg/kg; Subgroup 3, NCTD at 3 mg/kg; and Subgroup 4, PTX at 60 mg/kg combined with NCTD at 3 mg/kg.