Palladacycles catalyse the oxidation of critical thiols of the mitochondrial membrane proteins and lead to mitochondrial permeabilization and cytochrome c release associated with apoptosis

Abstract

Permeabilization of the mitochondrial membrane has been extensively associated with necrotic and apoptotic cell death. Similarly to what had been previously observed for B16F10-Nex2 murine melanoma cells, PdC (palladacycle compounds) obtained from the reaction of dmpa (N,N-dimethyl-1-phenethylamine) with the dppe [1,2-ethanebis(diphenylphosphine)] were able to induce apoptosis in HTC (hepatoma, tissue culture) cells, presenting anticancer activity in vitro. To elucidate cell site-specific actions of dmpa:dppe that could respond to the induction of apoptosis in cancer cells in the present study, we investigated the effects of PdC on isolated RLM (rat liver mitochondria). Our results showed that these palladacycles are able to induce a Ca2+-independent mitochondrial swelling that was not inhibited by ADP, Mg2+ and antioxidants. However, the PdC-induced mitochondrial permeabilization was partially prevented by pre-incubation with CsA (cyclosporin A), NEM (N-ethylmaleimide) and bongkreic acid and totally prevented by DTT (dithiothreitol). A decrease in the content of reduced thiol groups of the mitochondrial membrane proteins was also observed, as well as the presence of membrane protein aggregates in SDS/PAGE without lipid and GSH oxidation. FTIR (Fourier-transform IR) analysis of PdC-treated RLM demonstrated the formation of disulfide bonds between critical thiols in mitochondrial membrane proteins. Associated with the mitochondrial permeabilization, PdC also induced the release of cytochrome c, which is sensitive to inhibition by DTT. Besides the contribution to clarify the pro-apoptotic mechanism of PdC, this study shows that the catalysis of specific protein thiol cross-linkage is enough to induce mitochondrial permeabilization and cytochrome c release.