Targeting the mutant BRAF protein is an accepted approach to the treatment of metastatic melanoma. Potent and specific BRAF inhibitors like vemurafenib and dabrafenib are superior to chemotherapy in treatment of BRAF mutant melanomas which represent nearly 50% of all melanomas. Previous studies have shown that certain isoforms of vitamin E and statins can have synergistic anti-cancer activity. We determined whether a combination of delta-tocotrienol (DT3), an unsaturated vitamin E isoform, and simvastatin, an HMG-CoA reductase inhibitor, can exert an anti-neoplastic activity on BRAF-mutated SK-MEL-28 and BRAF-wild type SK-MEL-2 melanoma cell lines and whether a differential effect would be evident.
MTS assays were used to analyze cytotoxicity. SK-MEL-28 and SK-MEL-2 cells were cultured in MEM media containing 10% serum and plated in 96-well culture plates for 24 hours then treated with DT3 (0-40 μM), simvastatin (0-5 μM), or a combination and dosed again at 48 hours. SK-MEL-28 and SK-MEL-2 cells grown in 60 mm plates and were treated with DT3 at concentrations of 40, 30, 20 μM, simvastatin at a concentrations of 20, 10, 5 μM or dissolution vehicle as a control for 6 h. Protein concentration of cell lysates was measured spectrophotometrically (GLO Max Multi+, Promega), using a BCA protein assay kit. The samples were run in SDS PAGE and blotted onto nitrocellulose membranes. Membranes were incubated with antibodies against Hsp 70 (Enzo Life Sciences, Farmingdale, NY), Hsp 90 (Santa Cruz, Dallas, TX), pS6 and pBAD (Cell Signaling, Danvers, MA).
Using MTS assay, we found that DT3 (IC50 38.8 μM) and simvastatin (IC50 22.7μM) have cytotoxic effects on melanoma cell line SK-MEL-28, but on the SK-MEL-2 cells DT3 does not have an effect at the concentrations studied (10-40 μM DT3) yet simvastatin (IC50 16.9 μM) does have cytotoxicity. Further studies determined that combinations of these drugs display a synergistic effect on SK-MEL-28 by inhibition of pS6 and pBAD and subsequent apoptosis. However, these effects are not observed in SK-MEL-2 cells; treated SK-MEL-2 cells show over-expression of Hsp70 and Hsp90 suggestive of a rescue effect leading to lesser cytotoxic activity. The selective cytotoxicity observed in BRAF-mutated cells and not in wild type BRAF melanoma cell lines by both DT3 and simvastatin warrants further research into the potential therapeutic use of these combinations. This observation has added importance in the light of recent findings that show the acquisition of BRAF mutation is an early event in melanogenesis and hence these compounds may have a key role in chemoprevention approaches to melanoma.
Citation Format: Kelley Cross, Victoria Palau, Marianne Brannon, Janet Lightner, Megan Dycus, William Stone, Koyamangalath Krishnan. Delta-tocotrienol and simvastatin induce cytotoxicity and synergy in BRAF mutant SK-MEL-28 but not in wild type BRAF SK-MEL-2 melanoma cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3568.