Cisplatin and Oxaliplatin Toxicity: Importance of Cochlear Kinetics as a Determinant for Ototoxicity

Background Cisplatin is a commonly used platinum anti-cancer drug. Regrettably cisplatin has dose-limiting ototoxic side effects, e.g. the drug can induce an irreversible hearing loss. The ototoxic mechanisms of cisplatin have not been elucidated in the human ear and no clinically useful oto-protectors are yet available. Cisplatin is a necessary part of many treatment regimes. Its beneficial therapeutic effects might be reduced if cisplatin was excluded from the treatment in order to protect the hearing function. In this work the ototoxic effects of cisplatin are studied with the aim to better understand the mechanisms behind the irreversible hearing loss induced by this drug. Oxaliplatin is a second generation platinum-derivative anti-cancer drug, free from ototoxic side effects in clinical practice. The effects of oxaliplatin on the inner ear have been studied in this work and the results are compared with cisplatin treatment. The two drugs differ regarding both anti-cancer effects and side effects, which could be attributed to differences in pharmacokinetic factors, cellular uptake and apoptotic mechanisms. The thioredoxin redox system with the enzyme thioredoxin reductase (TrxR) was studied in cochleae due to a suggested DNA-independent apoptotic mechanism of the hair cells. The cochlear pharmacokinetics of cisplatin was assessed and the transport protein organic cation transporter 2 (OCT2) was studied in relation to the ototoxic effect of cisplatin. Material and methods Cultured human colon carcinoma cells and cell cultures of rat organ of Corti were used for apoptosis studies in vitro following exposure to cisplatin and oxaliplatin. Cisplatin and oxaliplatin were administered i.v. to guinea pigs, followed by in vivo sampling of blood, cerebrospinal fluid (CSF) and scala tympani (ST) perilymph. Liquid chromatography with post-column derivatization was used to determine the concentration of parent drug in the samples. Electrophysiological hearing thresholds and the loss of hair cells were assessed to evaluate their ototoxic effects. Phenformin, a potential blocker of OCT2 was administered and the ototoxic side effect of cisplatin was evaluated. For immunohistochemical studies, cochlea from rat, guinea pig and pig were used, where TrxR and OCT2 were evaluated in the cochlea. TrxR-assays were used to measure the TrxR activity in cochlear tissue, both in vivo and in vitro. Results The results from the in vitro studies showed that addition of either cisplatin or oxaliplatin to the culture medium in organ of Corti cell cultures caused a similar amount of outer hair cell loss and inhibition of TrxR activity. Cisplatin exposure to cultured human colon carcinoma cells also reduced the activity of TrxR. The results from the in vivo studies showed that a considerable concentration of cisplatin was present in ST perilymph as compared with weak concentrations of oxaliplatin after high dose oxaliplatin i.v. Ten minutes after cisplatin administration, its concentration in ST perilymph was 4-fold higher in the basal turn of the cochlea as compared to the apex. Cisplatin could be analysed in ST perilymph for up to 120 min. Phenformin i.v. did not reduce the ototoxic side-effect of cisplatin. Positive immunoreactivity to TrxR was evident in both hair cells and spiral ganglion cells. Futhermore, OCT2 was expressed in the supporting cells of organ of Corti and in the spiral ganglion cells. Conclusion The transport of cisplatin to the vulnerable cells of hearing seems to be of major importance for the ototoxic effects. An early high concentration of cisplatin in the base of the cochlea and delayed elimination of cisplatin from ST perilymph may be related to the cisplatin-induced loss of outer hair cells in the basal turn of the cochlea. Cisplatin and oxaliplatin both cause similar ototoxic effects when the organ of Corti is directly exposed in vitro. The thioredoxin redox system with the TrxR enzyme may well play a critical role in cisplatininduced ototoxicity. The presence of OCT2 in the supporting cells indicates that this transport protein is primarily not involved in the uptake of cisplatin from the systemic circulation but rather from the deeper compartments of the cochlea. The knowledge elicited in this work will hopefully suggest objectives for further studies in order to develop oto-protective treatments to preserve the hearing of cisplatin treated patients

Cell Death by SecTRAPs: Thioredoxin Reductase as a Prooxidant Killer of Cells

BACKGROUND: SecTRAPs (selenium compromised thioredoxin reductase-derived apoptotic proteins) can be formed from the selenoprotein thioredoxin reductase (TrxR) by targeting of its selenocysteine (Sec) residue with electrophiles, or by its removal through C-terminal truncation. SecTRAPs are devoid of thioredoxin reductase activity but can induce rapid cell death in cultured cancer cell lines by a gain of function. PRINCIPAL FINDINGS: Both human and rat SecTRAPs killed human A549 and HeLa cells. The cell death displayed both apoptotic and necrotic features. It did not require novel protein synthesis nor did it show extensive nuclear fragmentation, but it was attenuated by use of caspase inhibitors. The redox active disulfide/dithiol motif in the N-terminal domain of TrxR had to be maintained for manifestation of SecTRAP cytotoxicity. Stopped-flow kinetics showed that NADPH can reduce the FAD moiety in SecTRAPs at similar rates as in native TrxR and purified SecTRAPs could maintain NADPH oxidase activity, which was accelerated by low molecular weight substrates such as juglone. In a cellular context, SecTRAPs triggered extensive formation of reactive oxygen species (ROS) and consequently antioxidants could protect against the cell killing by SecTRAPs. CONCLUSIONS: We conclude that formation of SecTRAPs could contribute to the cytotoxicity seen upon exposure of cells to electrophilic agents targeting TrxR. SecTRAPs are prooxidant killers of cells, triggering mechanisms beyond those of a mere loss of thioredoxin reductase activity

The structure of human thioredoxin reductase 1 provides insights into C-terminal rearrangements during catalysis

Human thioredoxin reductase (hTrxR) is a homodimeric flavoprotein crucially involved in the regulation of cellular redox reactions, growth and differentiation. The enzyme contains a selenocysteine residue at its C-terminal active site that is essential for catalysis. This redox center is located on a flexible arm, solvent-exposed and reactive towards electrophilic inhibitors, thus representing a target for antitumor drug development. During catalysis reducing equivalents are transferred from the cofactor NADPH to FAD, then to the N-terminal active site cysteine residues and from there to the flexible C-terminal part of the other subunit to be finally delivered to a variety of second substrates at the molecule's surface. Here we report the first crystal structure of hTrxR1 (Sec→Cys) in complex with FAD and NADP+ at a resolution of 2.8 Å. From the crystals three different conformations of the carboxy-terminal arm could be deduced. The predicted movement of the arm is facilitated by the concerted action of the three side-chain residues of N418, N419 and W407, which act as a guiding bar for the C-terminal sliding process. As supported by previous kinetic data, the three visualized conformations might reflect different stages in enzymatic catalysis. Comparison with other disulfide reductases including human glutathione reductase revealed specific inhibitor binding sites in the intersubunit cavity of hTrxR that can be exploited for structure-based inhibitor development

Truncated mutants of human thioredoxin reductase 1 do not exhibit glutathione reductase activity

The substrate spectrum of human thioredoxin reductase (hTrxR) is attributed to its C-terminal extension of 16 amino acids carrying a selenocysteine residue. The concept of an evolutionary link between thioredoxin reductase and glutathione reductase (GR) is presently discussed and supported by the fact that almost all residues at catalytic and substrate recognition sites are identical. Here, we addressed the question if a deletion of the C-terminal part of TrxR leads to recognition of glutathione disulfide (GSSG), the substrate of GR. We introduced mutations at the putative substrate binding site to enhance GSSG binding and turnover. However, none of these enzyme species accepted GSSG as substrate better than the full length cysteine mutant of TrxR, excluding a role of the C-terminal extension in preventing GSSG binding. Furthermore, we show that GSSG binding at the N-terminal active site of TrxR is electrostatically disfavoured

Gold(I)-Phosphinkomplexe für die irreversible Hemmung von humanen Disulfid-Reduktasen

Redox-basierte Chemotherapie: Die irreversible Hemmung der humanen Glutathion-Reduktase resultiert in einem einmaligen S-AuI-S-Strukturmotiv, das kristallographisch charakterisiert wurde. Eine kinetische Analyse der schnellen und effektiven Hemmung von Redoxenzymen sowie DNA-Wechselwirkungsstudien, ergänzt durch Zellproliferationstests, belegen das chemotherapeutische Potenzial von Phosphinmetallkomplexe