Targeting of redox-controlling proteins of the thioredoxin family by oxaliplatin

2345

The antiproliferative effects of platinum drugs are usually ascribed to Pt-DNA adducts. However, protein-Pt adducts, which are an order of magnitude more numerous than DNA-Pt adducts, might be an additional factor that contributes to apoptosis by distorting protein redox homeostasis. The redox status of cellular proteins is mainly controlled by the antiapoptotic thioredoxin (Trx) and its regenerating enzyme thioredoxin reductase (TrxR). The Trx system was implicated in the sensitivity/resistance to cisplatin. Our previous studies with the third generation platinum drug oxaliplatin suggested that this drug in particular may exert effects on targets other than DNA. For example, oxaliplatin needs to form only half of DNA-Pt adducts formed by cisplatin for equitoxic effects. The objective of this study was to examine the potential of oxaliplatin to target the Trx system. The results demonstrate that oxaliplatin inhibits the activity of purified human Trx with the IC₅₀ of ∼20 μM after a 60 min preincubation of the enzyme with the drug. Purified mammalian TrxR is significantly inhibited even without preincubation with 50 μM oxaliplatin producing ∼40% inhibition. Even a brief (10 min) preincubation, however, enhances the inhibition (from 6 to 26% for 10 μM drug). The need for preincubation in these determinations is consistent with the putative covalent binding of oxaliplatin to target proteins. Atomic absorption determinations are under way to corroborate whether the inhibition of the enzymatic activities of Trx and TrxR is indeed accompanied by the formation of protein-Pt adducts. The effects of oxaliplatin on the proteins of the Trx system are specific as the drug did not inhibit the activity of glutathione S-transferase (GST), either when added directly to the reaction or after a 60 min preincubation. To assess the effects of oxaliplatin on the cellular activities of Trx and TrxR, prostate cancer LNCaP-Pro5 cells were exposed to oxaliplatin for 24 h. Cellular extracts from these cells were next used in the respective enzymatic assays. The effects of oxaliplatin on the intracellular activity of Trx were modest with 25 μM drug producing ∼30% inhibition and 10 μM drug showing no significant effect. However, markedly more potent inhibition was observed in case of TrxR with the IC₅₀ of ∼10 μM and a near complete inhibition ( ∼85%) at 25 μM oxaliplatin. These concentrations are comparable to those inducing apoptosis in LNCaP-Pro5 cells. Overall, the results demonstrate that oxaliplatin affects the key members of the system controlling protein redox status. The drug is clearly more inhibitory against TrxR, although even a modest effect on Trx is likely to contribute in a cumulative fashion to the overall impairment of the Trx system. These findings strongly support the notion that the distortion of protein redox regulation can be a factor in the antiproliferative and proapoptotic activities of oxaliplatin. (Supported by CA112175 and CA80936).