Inhibition of the thioredoxin system is a basis for the antileukemic potential of 13-hydroxy-15-oxo-zoapatlin.

The mammalian thioredoxin (Trx) system, composed of Trx, Trx reductase (TrxR), and NADPH, is the most important thiol system involved in the redox control of signaling and regulatory proteins in apoptosis and cell proliferation. Here we addressed the inhibition of the Trx system by 13-hydroxy-15-oxo-zoapatlin (OZ), a nor-kaurane diterpene previously shown to possess proapoptotic potential and to cause cell cycle arrest in leukemia cells. OZ was found, by both biochemical and mass spectrometry-based approaches, to target Trx1 and TrxR in a cell-free system. In particular, the formation of reversible OZ adducts to Trx1 Cys35, Cys62, and Cys73 was demonstrated. We next showed that OZ efficiently inhibited Trx and TrxR catalytic activity in Molt4 cells. The occurrence of oxidative modifications of Trx molecules was assessed by "redox Western blot" analyses. OZ-mediated Trx oxidation resulted in apoptosis signaling kinase-1 release and activation of downstream JNK and p38 pathways. By means of specific inhibitors of these two stress-activated protein kinases, we demonstrated that the JNK pathway plays a major role in determining the apoptotic fate of OZ-exposed cells, whereas p38 activation seems to be involved mainly in OZ-induced G2/M block.