Transient activation of EGFR/AKT cell survival pathway and expression of survivin contribute to reduced sensitivity of human melanoma cells to betulinic acid

Int J Oncol. 2005 Sep;27(3):823-30.

Abstract

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.

Publication types

  • Comparative Study
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Antineoplastic Agents, Phytogenic / pharmacology
  • Blotting, Western
  • Cell Line
  • Cell Line, Tumor
  • Cell Survival / drug effects
  • Cells, Cultured
  • Drug Resistance, Neoplasm
  • Drug Synergism
  • Embryo, Mammalian / cytology
  • Epidermal Growth Factor / pharmacology
  • ErbB Receptors / antagonists & inhibitors
  • ErbB Receptors / genetics
  • ErbB Receptors / metabolism*
  • Fibroblasts / cytology
  • Fibroblasts / drug effects
  • Fibroblasts / metabolism
  • Humans
  • Inhibitor of Apoptosis Proteins
  • Keratinocytes / cytology
  • Keratinocytes / drug effects
  • Keratinocytes / metabolism
  • Melanoma / metabolism
  • Melanoma / pathology
  • Mice
  • Mice, Knockout
  • Microtubule-Associated Proteins / metabolism*
  • Mitogen-Activated Protein Kinase 1 / metabolism
  • Mitogen-Activated Protein Kinase 3 / metabolism
  • Neoplasm Proteins / metabolism*
  • Phosphorylation / drug effects
  • Protein-Serine-Threonine Kinases / metabolism*
  • Proto-Oncogene Proteins / metabolism*
  • Proto-Oncogene Proteins c-akt
  • Quinazolines / pharmacology
  • Survivin
  • Time Factors
  • Triterpenes / pharmacology*

Substances

  • Antineoplastic Agents, Phytogenic
  • BIRC5 protein, human
  • Inhibitor of Apoptosis Proteins
  • Microtubule-Associated Proteins
  • Neoplasm Proteins
  • Proto-Oncogene Proteins
  • Quinazolines
  • Survivin
  • Triterpenes
  • betulinic acid
  • Epidermal Growth Factor
  • ErbB Receptors
  • AKT1 protein, human
  • Protein-Serine-Threonine Kinases
  • Proto-Oncogene Proteins c-akt
  • Mitogen-Activated Protein Kinase 1
  • Mitogen-Activated Protein Kinase 3
  • 4-((3-bromophenyl)amino)-6,7-dimethoxyquinazoline