We found differences between the apoptosis signaling induced by cisplatin and oxaliplatin in proliferating HCT116 cancer cells, with redox-related effects being clearly associated with apoptosis induced by cisplatin but not by oxaliplatin under comparable conditions. Although these agents are sometimes regarded as interchangeable, these results show that they differ. We also identified thioredoxin reductase 1 as a potential target for cisplatin, in particular, in both HCT116 cells and cochlea. However, the differences in cochlear kinetics and cellular uptake that we found in the hearing end organ are sufficient to explain the difference in ototoxicity between cisplatin and oxaliplatin.
We have previously demonstrated that cisplatin has pronounced DNA damage- and nucleus-independent effects (13
). In addition, levels of cytosolic Ca2+
and reactive oxygen species increase by 50% within 1 hour after cisplatin treatment (38
), which is well before the onset of p53 accumulation, which occurs approximately 3 hours after treatment with either drug (M Berndtsson PhD, MSc E Hernlund MSc, Shoshan M, PhD; unpublished observations). In this study, we show that, although cisplatin and oxaliplatin induce similar levels of apoptosis in cultured HCT116 colon carcinoma cells, superoxide anion production and increased levels of intracellular calcium are required for cisplatin-induced but not oxaliplatin-induced apoptosis. Superoxide anion production was furthermore independent of DNA damage, as shown by the effects of cisplatin in enucleated cells. DNA-damaging drugs typically act by interfering with DNA replication (39
). However, cells in the organ of Corti are terminally differentiated and do not divide. Therefore, the apoptotic redox signaling induced by cisplatin in enucleated cells may be a possible mechanism of cisplatin-induced ototoxicity. It should also be noted that the chloride concentrations in the McCoy's culture medium used for the tumor cells and in the mammalian perilymph were in the same range (120 and 110–140 mM, respectively). Different extracellular chloride concentrations are thereby not likely to affect the cisplatin toxicity differently in cell culture and in the outer hair cells in the organ of Corti.
The involvement of calcium cations and superoxide anions in cisplatin-induced apoptosis but not oxaliplatin-induced apoptosis is in agreement with the observation that inhibition of thioredoxin reductase may affect calcium homeostasis (40
). Apoptosis was also induced by cisplatin despite only partial inhibition of thioredoxin reductase (), which might reflect the fact that cisplatin-derivatized thioredoxin reductase has not only lost thioredoxin-reducing activity but also gained a dominant function as pro-oxidant inducer of cell death (27
). This gain of function was associated with the conversion of thioredoxin reductase from an oxidoreductase with antioxidant activity to a SecTRAP with potent superoxide-producing NADPH oxidase capacity after its derivatization with cisplatin (27
). If SecTRAPs are formed in cisplatin-treated cells, then this mechanism may be involved in intracellular superoxide production, in prooxidant cell death induction, and also in the antiapoptotic effect observed after treatment with the superoxide scavanger Tiron. Because of the importance of thioredoxin reductase as an enzymatic target contributing to cisplatin toxicity (20
), because of the weaker inhibitory effect of oxaliplatin on its cellular activity, and because of the finding that thioredoxin reductase was expressed in the cochlea, we propose that the targeting of thioredoxin reductase by cisplatin could contribute to the ototoxicity of cisplatin, but this possibility must be investigated further.
We have also shown that the lack of major cochlear injury during treatment with oxaliplatin is likely explained by the pharmacokinetic profile of oxaliplatin. There was a striking difference between cisplatin and oxaliplatin in the uptakes into scala tympani perilymph fluid and in total platinum concentrations in cochlear cells. In the inner ear, transport of drugs between blood and receptor cells or neural tissue is hindered by the presence of barrier cell layers. In analogy with the blood–brain barrier, the concept of a blood–labyrinth barrier is generally accepted. This barrier can roughly be divided into the blood–perilymph barrier and the perilymph–endolymph barrier, although the functional division between these compartments is not fully understood. The blood–perilymph barrier, which consists mainly of endothelial cells sealed by tight junctions, is most likely the major barrier for pharmacologic substances in reaching the sensory epithelium of the hearing and balance organs. Cisplatin-induced ototoxicity is known to be mediated via injury to several terminally differentiated cellular targets in the cochlea (42
), including the marginal and intermediate cells of the stria vascularis (43
), which is a major component of the perilymph–endolymph barrier.
Our study had several limitations. First, we discovered more pronounced oxidative stress and calcium involvement in the cytotoxicity of cisplatin than of oxaliplatin in cell cultures, but it is not clear if these differences mediate differences in cytotoxicity also in vivo. Second, we identified thioredoxin reductase as a plausible cisplatin target in the cochlea but could present no absolute evidence for its involvement in cochlear ototoxicity. This task, which is highly demanding methodologically, is beyond the scope of the present work but is a major goal of ongoing endeavors. Third, it should be noted that it is technically not possible to evaluate the scala tympani perilymph concentration time curves of cisplatin and oxaliplatin for the individual guinea pigs, because only one sample can be taken from each cochlea by aspiration procedures. The statistical technique presented by Yuan (37
) that we used in this analysis is a valuable tool for construction of concentration–time curves and for calculation of AUC from a group of guinea pigs in which only one sample was collected from each cochlea. The technique also allows estimation of the variance of AUC in such experiments. However, in this analysis, we determined the scala tympani perilymph concentrations separately in the left and right cochlea to reduce the number of experimental guinea pigs. The statistical test used for comparison of the AUC for cisplatin and oxaliplatin can thereby be viewed as an estimation procedure, as the number of observations was low. However, our conclusions were further supported by the observed fairly large concentration differences between cisplatin and oxaliplatin, as illustrated in . Thus, limited cochlear uptake of oxaliplatin is a major explanation for the lower ototoxicity of oxaliplatin than cisplatin. These in vivo experiments were performed in guinea pigs, and it should not be taken for granted that human subjects have the same cochlear pharmacokinetics. For technical and ethical reasons, it is impossible to repeat our experiments in human subjects. We do believe, though, that the major aspects of our conclusions are valid for humans.
The AUC of oxaliplatin in the guinea pig perilymph was only half of that of cisplatin when equimolar doses of drugs were administered. It should also be noted that the therapeutic doses of oxaliplatin and cisplatin in humans are equimolar (ie, 135 mg/m2, or 0.34 mmol/m2, and 100 mg/m2, or 0.33 mmol/m2, respectively). Thus, our findings may represent differences in the transport of the two drugs from the blood to the extracellular compartments of the cochlea. We can hypothesize that four mechanisms may, in various possible combinations, be responsible for the higher cochlear concentration of cisplatin as reflected by perilymph kinetics. First, the elimination half-life of cisplatin was longer than that of oxaliplatin. One would thereby expect a higher concentration of cisplatin in the blood for a longer period, which would favor its uptake in scala tympani perilymph. Second, the permeability of the blood–labyrinth barrier might favor influx of cisplatin. Third, increased influx of cisplatin to the cochlear extracellular fluids might also be provided by a facilitated influx transport (eg, via a single or a group of organic cationic transporters). Fourth, oxaliplatin might also have a greater efflux from the scala tympani perilymph than cisplatin.
At equimolar doses, cisplatin (12.5 mg/kg), as expected, had severe ototoxic effects in the guinea pig model, whereas oxaliplatin (16.6 mg/kg) had minimal ototoxic effects. Reducing the dose of cisplatin to 5 mg/kg resulted in a low perilymph concentration of drug similar to that of oxaliplatin at 16.6 mg/kg and consequently did not induce the drug-induced auditory brain stem threshold shift and injury to the outer hair cells. These results strongly indicate that the pharmacokinetic differences between cisplatin and oxaliplatin may be sufficient to explain their different ototoxic profiles.