Betulinic acid-induced Mcl-1 expression in human melanoma--mode of action and functional significance.
BACKGROUND
Currently there is no information on the regulation of expression and physiological role of the anti-apoptotic protein Mcl-1 in cells of the melanocytic lineage. This study investigates the regulation and expression of Mcl-1 in human melanoma cells, which was recently found to be induced by betulinic acid, a compound with anti-melanoma and apoptosis-inducing potential.


METHODS
Mcl-1 phosphorthioate antisense oligonucleotides were used to investigate the effect of downregulating the expression of Mcl-1. Regulation of Mcl-1 expression was analyzed with the specific PI3-kinase inhibitors LY294002 and wortmannin and the inhibitor of MAP-kinase activation, PD98059. Western blot analysis was performed with anti ERK1/2, Mcl-1, Bak, Bcl-x and Bax antibodies. Activation status of PI-3 kinase and MAP-kinase pathways was investigated using phospho-Akt and phosphorylation-state independent Akt as well as phospho-MAP kinase, phospho-MEK and phospho-GSK-3alpha/beta antibodies.


RESULTS
Upregulation of Mcl-1 in human melanoma cells by betulinic acid is mediated via a signal-transduction pathway that is inhibited by LY294002 and wortmannin. Betulinic acid-induced phosphorylation and activation of the Akt protein kinase was inhibited by LY294002. The inhibitor PD98059 reduced expression levels of Mcl-1 in melanoma cells and this effect was counteracted by betulinic acid. Downregulation of Mcl-1 by antisense oligodeoxynucleotides in combination with betulinic treatment led to a synergistic effect regarding growth inhibition.


CONCLUSIONS
These results suggest that in human melanoma cells Mcl-1 is (i) of functional relevance for survival and (ii) subject to dual regulation by the MAP- kinase pathway and a pathway involving protein kinase B/Akt, the latter of which is modulated in response to betulinic acid. This study provides an experimental foundation for future therapeutic strategies using anti-Mcl-1 antisense oligonucleotides in human melanoma.
Selzer
Edgar
E
Department of Clinical Pharmacology, Section of Experimental Oncology and Molecular Pharmacology, University Hospital Vienna, Austria. Edgar.Selzer@AKH-Wien.ac.at
Thallinger
Christiane
C
Hoeller
Christoph
C
Oberkleiner
Philipp
P
Wacheck
Volker
V
Pehamberger
Hubert
H
Jansen
Burkhard
B
D000730:Androstadienes / Q000494:pharmacology; D018929:Cell Culture Techniques; D002867:Chromones / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D008545:Melanoma / Q000378:metabolism*; D009025:Morpholines / Q000494:pharmacology; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000235:genetics*; D053978:Pentacyclic Triterpenes; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D014315:Triterpenes / Q000378:metabolism*; D000077191:Wortmannin; D000094062:Betulinic Acid
D000730:Androstadienes; D002867:Chromones; D004791:Enzyme Inhibitors; D009025:Morpholines; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D009363:Neoplasm Proteins; D053978:Pentacyclic Triterpenes; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D014315:Triterpenes; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C494918:AKT1 protein, human; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D000077191:Wortmannin; D000094062:Betulinic Acid