Effects of betulinic acid alone and in combination with irradiation in human melanoma cells

Recently, betulinic acid was identified as a highly selective inhibitor of human melanoma growth and was reported to induce apoptosis in these cells. We have investigated the growth-inhibitory properties of this compound alon e and in combination with ionizing radiation in a panel of established huma n melanoma cell lines as well as in normal human melanocytes. Betulinic aci d strongly and consistently suppressed the growth and colony-forming abilit y of all human melanoma cell lines investigated. In combination with ionizi ng radiation the effect of betulinic acid on growth inhibition was additive in colony-forming assays. Betulinic acid also induced apoptosis in human m elanoma cells as demonstrated by Annexin V binding and by the emergence of cells with apoptotic morphology. The growth-inhibitory action of betulinic acid was more pronounced in human melanoma cell lines than in normal human melanocytes. Notably, despite the induction of apoptosis, analysis of the e xpression of Bcl-2 family members in betulinic-acid-treated cells revealed that expression of the anti-apoptotic protein Mcl-1 was induced. Furthermor e, the antiproliferative action of betulinic acid seemed to be independent of the p53 status. The properties of betulinic acid make it an interesting candidate, not only as a single agent but also in combination with radiothe rapy. We conclude that the strictly additive mode of growth inhibition in c ombination with irradiation suggests that the two treatment modalities may function by inducing different cell death pathways or by affecting differen t target cell populations.