Carcinogenesis Advance Access published online on April 30, 2009
Carcinogenesis, doi:10.1093/carcin/bgp105
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The isatin-Schiff base copper(II) complex Cu(isaepy)2 acts as delocalized lipophilic cation, yields widespread mitochondrial oxidative damage and induces AMP-activated protein kinase-dependent apoptosis
1 Department of Biology, University of Rome "Tor Vergata", via della Ricerca Scientifica, 00133 Rome, Italy
2 Departamento de Química Fundamental, Instituto de Química, Universidade de Sâo Paulo, P.O. Box 26077, CEP 05513-970, Sâo Paulo, SP, Brazil
3 Research Centre IRCCS San Raffaele Pisana, Via dei Bonacolsi, 00163, Rome, Italy
To whom correspondence should be addressed: Maria R. Ciriolo, Department of Biology, University of Rome "Tor Vergata", Via della Ricerca Scientifica, 00133 Rome, Italy. Phone: +39 06 7259 4369, Fax: +39 06 7259 4311, E-mail: ciriolo{at}bio.uniroma2.it
We previously demonstrated that Bis[(2-oxindol-3-ylimino)-2-(2-aminoethyl)pyridine-N,N’]copper(II) (Cu(isaepy)2) was an efficient inducer of the apoptotic mitochondrial pathway. Here, we deeply dissect the mechanisms underlying the ability of Cu(isaepy)2 to cause mitochondriotoxicity. In particular, we demonstrate that Cu(isaepy)2 increases NADH-dependent oxygen consumption of isolated mitochondria and that this phenomenon is associated with oxy-radical production and insensitive to ADP. These data indicate that Cu(isaepy)2 behaves as an uncoupler and this property is also confirmed in cell systems. Particularly, SH-SY5Y cells show: i) an early loss of mitochondrial trans-membrane potential; ii) a decrease in the expression levels of respiratory Complex components; iii) a significant ATP decrement. The causative energetic impairment mediated by Cu(isaepy)2 in apoptosis is confirmed by experiments carried out with 
0 cells, or by glucose supplementation, where cell death is significantly inhibited. Moreover, gastric and cervix carcinoma AGS and HeLa cells, which rely most of their ATP production on oxidative phosphorylation, show a marked sensitivity towards Cu(isaepy)2. AMP-activated protein kinase (AMPK), which is activated by events increasing the AMP/ATP ratio, is deeply involved in the apoptotic process, because the over-expression of its dominant/negative form completely abolishes cell death. Upon glucose supplementation AMPK is not activated, confirming its role as fuel-sensing enzyme that positively responds to Cu(isaepy)2-mediated energetic impairment by committing cells to apoptosis. Overall, data obtained indicate that Cu(isaepy)2 behaves as DLC and induces mitochondrial-sited ROS production. This event results in mitochondrial dysfunction and ATP decrease, which in turn triggers AMPK-dependent apoptosis.
Received January 15, 2009; revised March 31, 2009; accepted April 25, 2009.