We evaluate how 3-acetylation modulates the in vitro activity of ursolic acid in melanoma cells alone or in combination treatments with quercetin. Anti-proliferative studies on A375 cells and adult human dermal fibroblasts included analyses on cell cycle distribution, caspase activity, phosphatidylserine translocation, cell morphology and Bax/Bcl-2 protein expression. Then, 2D and 3D migration of B16F10 cells were studied using scratch and Transwell assays, respectively. Ursolic acid and 3-O-acetylursolic acid have shown similar GI₅₀ on A375 cells (26 µM vs. 32 µM, respectively) significantly increased both early and late apoptotic populations, activated caspases 3/7 (48–72 h), and enhanced Bax whilst attenuating Bcl-2 expression. Ursolic acid caused elevation of the sub-G1 population whilst its 3-acetyl derivative arrested cell cycle at S phase and induced strong morphological changes. Combination treatments showed that ursolic acid and quercetin act synergistically in migration assays but not against cell proliferation. In summary, 3-O-acetylursolic acid maintains the potency and overall apoptotic mechanism of the parent molecule with a more aggressive influence on the morphology of A375 melanoma cells but the 3-acetylation suppresses its anti-migratory properties. We also found that ursolic acid can act in synergy with quercetin to reduce cell migration.

Ferruginol is a promising abietane-type antitumor diterpene able to induce apoptosis in SK-Mel-28 human malignant melanoma. We aim to increase this activity by testing the effect of a small library of ferruginol analogues. After a screening of their antiproliferative activity (SRB staining, 48 h) on SK-Mel-28 cells the analogue 18-aminoferruginol (GI50 ≈ 10 µM) was further selected for mechanistic studies including induction of apoptosis (DAPI staining, p < 0.001), changes in cell morphology associated with the treatment (cell shrinkage and membrane blebbing), induction of caspase-3/7 activity (2.5 at 48 h, 6.5 at 72 h; p < 0.0001), changes in the mitochondrial membrane potential (not significant) and in vitro effects on cell migration and cell invasion (Transwell assays, not significant). The results were compared to those of the parent molecule (ferruginol, GI50 ≈ 50 µM, depolarisation of mitochondrial membrane p < 0.01 at 72 h; no caspases 3/7 activation) and paclitaxel (GI50 ≈ 10 nM; caspases 3/7 activation p < 0.0001) as a reference drug. Computational studies of the antiproliferative activity of 18-aminoferruginol show a consistent improvement in the activity over ferruginol across a vast majority of cancer cells in the NCI60 panel. In conclusion, we demonstrate here that the derivatisation of ferruginol into 18-aminoferruginol increases its antiproliferative activity five times in SK-MEL-28 cells and changes the apoptotic mechanism of its parent molecule, ferruginol.

Ferruginol is a promising abietane-type antitumor diterpene able to induce apoptosis in SK-Mel-28 human malignant melanoma. We here aim to increase this activity by testing the effect of a small library of ferruginol analogues. After an screening of their antiproliferative activity (SRB staining) on SK-Mel-28 cells the analogue 18-aminoferruginol (GI50=10 µM) was further selected for mechanistic studies including effects on mitochondrial viability (MTT assay, IC50=10 µM p<0.0001), induction of apoptosis (DAPI staining, p<0.001), changes in cell morphology associated with the treatment (cell shrinkage and membrane blebbing), induction of caspase-3/7 activity (2.5x at 48h, 6.5x at 72h; p<0.0001), changes in the mitochondrial membrane potential (not significant), and effects on 2D cell migration and 3D cell invasion (modified Boyden assay, not significant). The results were compared to those of the parent molecule (ferruginol, GI50»50 µM) and paclitaxel (GI50=10 nM) as a reference drug. In conclusion, we demonstrate here that derivatization of ferruginol into 18-aminoferruginol increases its antiproliferative activity 5 times in SK-MEL-28 cells as well as changing the apoptotic mechanism of its parent molecule ferruginol from activation of caspases without depolarization of mitochondrial membrane.