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Search for selective modulators of human caspase 3 and 7, using a
yeast expression system
I. Coutinho 1a , M. Neves 2a , C. Baptista 1a , H. Cidade 2a , M. Pinto 2a , M.S.J. Nascimento 2b , M.
Côrte-Real 3 , J. Gonçalves 1b and L. Saraiva 1a
1 REQUIMTE/CEQUP, Department of Microbiology a and Pharmacology b , Faculty of Pharmacy,
University of Porto, Portugal
2 CEQUIMED-UP, Department of Organic Chemistry a and Microbiology b , Faculty of Pharmacy,
University of Porto, Portugal
3 Molecular and Environmental Biology Centre, University of Minho, Portugal
Caspase 3 and 7 are members of the cysteine protease family with a crucial role in the
execution of apoptosis, therefore representing promising pharmacological targets in anticancer
therapy [1]. Several flavonoids, such as 3,7-dihydroxyflavone and baicalein, showed to inhibit
cell survival by activation of a caspase-dependent apoptotic pathway [2]. Furthermore, it was
suggested that prenyl side chains increase flavonoids activities, namely the antiproliferative
effect against different human tumour cell lines [3].
Saccharomyces cerevisiae has been used by our group to express individual caspase family
members in order to search for selective modulators of these proteins. As described for human
cells [1], we confirmed, by cell viability assays, analysis of plasma membrane integrity, DNA
fragmentation and reactive oxygen species production, that expression of human caspase 3 or 7
in yeast causes a marked growth inhibition associated with the induction of an apoptotic cell
death. Based on this, activators and inhibitors of caspase 3 and 7 will enhance and decrease,
respectively, caspase-induced cell death, without interfering with control yeast (without
expression of human caspase). Using this yeast phenotypic assay, we analysed the effect of
several flavonoid derivatives obtained by CEQUIMED-UP group, baicalein (1), 3,7dihydroxyflavone
(2), 7-prenylbaicalein (3), 7-geranylbaicalein (4), 3-hydroxy-7prenyloxyflavone
(5), 3-hydroxy-7-geranyloxyflavone (6) and artelastin (7), with cytotoxic
effect on human tumour cell lines.
The results obtained revealed a differential modulation of caspase 3 and 7 by these flavonoids.
Compounds 2, 4 and 5 behaved as selective activators of caspase 3, while compounds 3, 6 and
7 behaved as selective activators of caspase 7. Compound 1 did not interfere with both
caspases. In conclusion, this work establishes a first line screening approach that contributes
for the search of new selective caspase 3 and 7 modulators in a more efficient way.
We thank to REQUIMTE, to CEQUIMED-UP (I&D 4040/2007) and to U.Porto/Santander
Totta for financial support. I. Coutinho (SFRH/BD/36066/2007) and M. Neves
(SFRH/BD/21770/2005) are recipient of a PhD fellowship from FCT.
References:
[1] Lavrik, I. et al. (2005), Caspases: pharmacological manipulation of cell death. J. Clin. Invest. 115
(10), 2665–2672.
[2] Monasterio, A. et al. (2004), Flavonoids induce apoptosis in human leukemia U937 cells through
caspase- and caspase-calpain-dependent pathways. Nutr. Cancer, 50 (1), 90-100.
[3] Epifano, F. et al. (2007), Chemistry and pharmacology of oxyprenylated secondary plant
metabolites, Phytochemistry, 68 (7), 939-953.
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