Carcinogenesis Advance Access originally published online on April 30, 2009
Carcinogenesis 2009 30(7):1115-1124; 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 Research Centre IRCCS San Raffaele Pisana, Via dei Bonacolsi, 00163 Rome, Italy
3 Departamento de Química Fundamental, Instituto de Química, Universidade de Sâo Paulo, PO Box 26077, CEP 05513-10 970, Sâo Paulo, SP, Brazil
* To whom correspondence should be addressed. Tel: +39 06 7259 4369; Fax: +39 06 7259 4311; Email: 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 adenosine diphosphate. 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 transmembrane potential; (ii) a decrease in the expression levels of respiratory complex components and (iii) a significant adenosine triphosphate (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 toward Cu(isaepy)2. Adenosine monophosphate-activated protein kinase (AMPK), which is activated by events increasing the adenosine monophosphate:ATP ratio, is deeply involved in the apoptotic process because the overexpression 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 delocalized lipophilic cation and induces mitochondrial-sited reactive oxygen species production. This event results in mitochondrial dysfunction and ATP decrease, which in turn triggers AMPK-dependent apoptosis.
Abbreviations: ADP, adenosine diphosphate; AMPK, adenosine monophosphate-activated protein kinase; ATP, adenosine triphosphate; DLC, delocalized lipophilic cation; ROS, reactive oxygen species
Received January 15, 2009; revised March 31, 2009; accepted April 25, 2009.