Betulinic acid (BA), a pentacyclic
triterpene first identified less than a decade ago, has served as a
melanoma-specific
cytotoxic agent, and yet its specificity is being challenged. Recently, we found that human
melanoma cells exhibited less sensitivity to
betulinic acid than human skin keratinocytes. This study was designed to investigate the cell signaling pathway leading human
melanoma cells to increased resistance to
betulinic acid treatment. In vitro experiments using cultured human
melanoma cells indicated that
betulinic acid transiently induced
survivin expression. The expression of
survivin started 30 min post-
betulinic acid treatment, peaked at 2 h, remained elevated for 8 h and returned to basal level within 24 h. Similarly, epithelial
growth factor (
EGF) treatment induced expression of
survivin in a time-dependent manner. Since epithelial
growth factor receptor (EGFR) activation leads to the activation of cell signaling components that are important to cell survival, we next examined whether BA-induced
survivin expression is mediated by the EGFR pathway. The results showed that BA induced EGFR
tyrosine phosphorylation in a time-dependent manner. Further, BA strongly induced AKT phosphorylation in a similar pattern. AKT activation started 15 min post-treatment, peaked at approximately 1 h, remained elevated for 4 h and returned to basal level within 8 h. BA also induced ERK activation and, in contrast, weakly induced JNK and p38 activation. Pretreatment of EGFR inhibitor
PD153035 blocked BA-induced EGFR phosphorylation, ERK and AKT activation, and
survivin expression. Results of the MTT
dye assay showed that a combination of
PD153035 and BA enhanced
melanoma cell death. Collectively, we conclude that
betulinic acid transiently activated the EGFR/AKT cell survival pathway and induced
survivin expression, contributing to less sensitivity in human
melanoma cells. The data suggest that a combination of the EGFR inhibitor and
betulinic acid may be a better clinical option to treat human
melanoma.