Targeting human melanoma growth with non-calcemic vitamin d3 hydroxyderivative: A synergistic approach with vemurafenib

Background: Melanoma remains a major clinical challenge due to its resistance to current targeted therapies, emphasizing the need for new preventive and therapeutic strategies. The vitamin D receptor (VDR) and its ligands are promising, low-toxicity targets. Our team has discovered an alternative, noncanonical vitamin D-metabolic pathway initiated by CYP11A1, generating hydroxyderivatives such as 20(OH)D₃ with antioxidative, photoprotective, and anticancer activities through VDR and other nuclear receptors (LXR, AhR, ROR). We hypothesize that 20(OH)D₃, alone or combined with vemurafenib (VEM), can inhibit melanoma tumorigenic behavior through VDR-dependent and independent mechanisms. Methods: In vitro studies used migration, CFU, and PrestoBlue viability assays on melanoma lines (WM3960, WM1366, WM3942, WBM550). Spheroid assays utilized 3D culture and TUNEL staining to assess apoptosis. In vivo efficacy was evaluated in nude mice implanted with either wild-type (WT) or VDR-knockout ( VDR ⁻/⁻ ) WM164 ( BRAF V600E ) melanoma cells. Mice received vehicle, 20(OH)D₃ VEM, or combination therapy three times weekly. Tumor growth and necrosis were monitored, and protein expression was analyzed by Western blotting. Results: Cell migration assays demonstrated that the treatment with novel vitamin D₃ derivatives markedly inhibited the migratory capacity of WM3960 PDX melanoma cells. CFUs further confirmed a significant reduction in melanoma cell growth across several melanoma cell lines. Viability assay revealed decreased fluorescence intensity in treated cells relative to controls, indicating reduced metabolic activity, viability, and proliferation. No apoptosis was detected using the TUNEL System, suggesting that the observed effects were primarily cytostatic rather than apoptotic. In vivo , WT WM164 xenografts treated with the combination of 20(OH)D₃ and VEM exhibited the strongest tumor suppression, followed by VEM monotherapy, whereas 20(OH)D₃ alone had minimal impact. In VDR ⁻/⁻ tumors, both VEM and combination treatments achieved comparable inhibition, indicating both VDR-dependent and independent mechanisms of action. Western blot confirmed synergistic downregulation of phospho AKT, ERK1/2, and NF-κB signaling pathways in combination-treated tumors, consistent with enhanced suppression of oncogenic signaling. Notably, male mice displayed greater sensitivity to the combination therapy compared with females. Conclusion: The non-calcemic vitamin D₃ derivative 20(OH)D₃, especially when combined with vemurafenib, inhibits human melanoma growth by blocking oncogenic signaling pathways, independent of apoptosis. Due to its natural origin, safety profile, and pathway selectivity, 20(OH)D₃ has strong potential as a preventive and therapeutic agent against melanoma progression and metastasis.