Betulinic Acid-induced Mcl-1 Expression in Human Melanoma — Mode of Action and Functional Significance

Edgar Selzer,Burkhard Jansen,Christiane Thallinger,Volker Wacheck,Philipp Oberkleiner,Hubert Pehamberger,Christoph Hoeller

doi:10.1007/bf03402094

Abstract

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. 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. 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. 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.

Highlights

Mcl-1 is a member of the Bcl-2 family, the existence of which was described for the first time only recently in cells of the melanocytic lineage [1,2]
We recently investigated the expression of Bcl-2 homologues after treatment with betulinic acid in melanocytes and found that betulinic acid maximally induced the expression of the anti-apoptotic protein Mcl-1 in melanoma cells within 6 to 8 hours
It is of interest to note in this context that this might be the first report of Bcl-w expression, a pro-apoptotic protein, in human melanoma cells

Summary

Introduction

Mcl-1 is a member of the Bcl-2 family, the existence of which was described for the first time only recently in cells of the melanocytic lineage [1,2]. Our group found that the expression of the anti-apoptotic protein Mcl-1 in human melanoma is induced rapidly by betulinic acid, a substance that induces apoptosis in a TP53 independent manner (1, and references therein). These observations led to further investigation into the function and regulation of expression of Mcl-1 in human melanocytes or melanoma cells. 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. 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-3␣/␤ antibodies

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