Lenalidomide exerts cytostatic activity on melanoma cells by negative regulation of their cell cycle

Background: Recurrence free survival in patients with metastatic melanoma (MM) is recently improved by targeted and immunotherapy regimens although the prognosis remains poor and requires further therapeutic efforts. Lenalidomide (Len) is an immunomodulant drug (IMiD) showing anti-inflammatory and anti-tumor properties in hematologic disorders as well as safety and tolerability in phase 2/3 clinical trials in MM. In addition it exerts anti-angiogenic effect once combined to sorafenib in the human ocular melanoma xenograft model. While it has been demonstrated that Len activates CD28 checkpoint expressed by T cells that drives the NF-kB activation downstream p21 cell cycle regulator and increases the T cell cytotoxicity by overcoming the inhibitory effect of CTLA4. Therefore, this research is aimed to investigate the potential role of Len in melanoma. Materials and methods: Three melanoma cell lines (A375, SKMEL28, WN266) were cultured for 24 hours in serum free medium to synchronize the cell cycle phases and then in complete medium supplemented with Len at different concentrations from 0.01 to 100 µM. A standard curve of cytotoxicity was completed to measure the IC50 whereas both viability and proliferation of melanoma cells was evaluated by MTT assay. The effect of Len on the cycle phases was investigated by measuring the DNA content by flow-cytometry after cell staining with propidium iodide (PI). Finally, real-time PCR explored the gene expression levels of p21. Results: Melanoma cells showed a 3-fold decrease of their proliferation extent by Len at 10 µM (640 ± 30 OD vs 1.915 ± 15 OD, p<0.05). However, the majority of cells were viable (>91%) although frozen at the G0-G1 phase of the cell-cycle (64 ± 10%) with respect to untreated cells (38 ± 4.6%). Percentage number of apoptotic cells was apparently similar (Len-treated: 0.7 ± 0.02 vs untreated: 1.7 ± 0.04%) as well as those undergoing to the S and G2-M phases (Len-treated: 35 ± 3 vs untreated: 60 ± 2%). Len-treated melanoma cells resulted up-regulated in p21 RNA content. Conclusions: In agreement with the antiproliferative activity on multiple myeloma cells, Len exerts cytostatic effect in melanoma cell lines through p21 up-regulation and potential CDK4/6 modulation. This provides the rationale for planning further pre-clinical investigations with IMiDs in combination with CDK4/6 inhibitors in MM.