The ototoxicity of cisplatin, a widely used chemotherapeutic agent, involves a number of mechanisms, including perturbation of redox status, increase in lipid peroxidation, and formation of DNA adducts. In this study, we demonstrate that cisplatin increased the early immediate release and de novo synthesis of proinflammatory cytokines, including TNF-a, IL-1b, and IL-6, through the activation of ERK and NF-kB in HEI-OC1 cells, which are conditionally immortalized cochlear cells that express hair cell markers. Both neutralization of proinflammatory cytokines and pharmacologic inhibition of ERK significantly attenuated the death of HEI-OC1 auditory cells caused by cisplatin and proinflammatory cytokines. We also observed a significant increase in the protein and mRNA levels of proinflammatory cytokines in both serum and cochleae of cisplatin-injected rats, which was suppressed by intraperitoneal injection of etanercept, an inhibitor of TNF-a. Immunohistochemical studies revealed that TNF-a expression was mainly located in the spiral ligament, spiral limbus, and the organ of Corti in the cochleae of cisplatin-injected rats. NF-kB protein expression, which overlapped with terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling-positive signal, was very strong in specific regions of the cochleae, including the organ of Corti, spiral ligament, and stria vascularis. These results indicate that proinflammatory cytokines, especially TNF-a, play a central role in the pathophysiology of sensory hair cell damage caused by cisplatin.

In our previous study, we clearly demonstrated the roles of pro-inflammatory cytokines, including tumor necrosis factor-␣, interleukin-1␤ (IL-1␤), and IL-6, and subsequent reactive oxygen species (ROS) generation on the pathogenesis of cisplatin ototoxicity in vitro and in vivo. ROS generation in cisplatin-treated HEI-OC1 auditory cells was also correlated with changing mitochondrial membrane potential. However, the roles of NADPH oxidase in cisplatin-induced ROS generation and ototoxicity have not been fully elucidated. Herein, immunohistochemical studies demonstrated that treatment of cisplatin induced the expression of NADPH oxidase isoforms NOX-1 and NOX-4 in HEI-OC1 auditory cells. Expression of mRNA for NOX-1, NOX-4, NOXO1, NOXA1, p47phox , and p67 phox was also increased. Inhibition of NADPH oxidase with diphenyleniodonium chloride or apocynin abolished ROS production and the subsequent apoptotic cell death in cisplatin-treated cells. Furthermore, suppression of NOX1 and NOX4 expression by small interfering RNA transfection markedly abolished the cytotoxicity and ROS generation by cisplatin. Together, our data suggest that ROS generated, in part, through the activation of NADPH oxidase plays an essential role in cisplatin ototoxicity.

Cisplatin is one of the most widely used and highly effective drug for the treatment of various solid tumors; however, it has dose-dependent side effects on the kidney, cochlear, and nerves. Nephrotoxicity is the most well-known and clinically important toxicity. Numerous studies have demonstrated that several mechanisms, including oxidative stress, DNA damage, and inflammatory responses, are closely associated with cisplatin-induced nephrotoxicity. Even though the establishment of cisplatin-induced nephrotoxicity can be alleviated by diuretics and pre-hydration of patients, the prevalence of cisplatin nephrotoxicity is still high, occurring in approximately one-third of patients who have undergone cisplatin therapy. Therefore it is imperative to develop treatments that will ameliorate cisplatin-nephrotoxicity. In this review, we discuss the mechanisms of cisplatin-induced renal toxicity and the new strategies for protecting the kidneys from the toxic effects without lowering the tumoricidal activity.

Cisplatin is a widely used chemotherapeutic agent for the treatment of various tumors. In addition to its antitumor activity, cisplatin affects normal cells and may induce adverse effects, such as ototoxicity, nephrotoxicity, and neuropathy. Various mechanisms, such as DNA adduct formation, mitochondrial dysfunction, oxidative stress, and inflammatory responses, are critically involved in cisplatin-induced adverse effects. As NAD+ is a cofactor for various enzymes associated with cellular homeostasis, we studied the effects of increased NAD+ levels by means of NAD(P)H:quinone oxidoreductase 1 (NQO1) activation using a known pharmacological activator (β-lapachone) in wild-type and NQO1−/− mice on cisplatin-induced renal dysfunction in vivo. The intracellular NAD+/NADH ratio in renal tissues was significantly increased in wild-type mice co-treated with cisplatin and β-lapachone compared with the ratio in mice treated with cisplatin alone. Inflammatory cytokines and biochemical markers for renal damage were significantly attenuated by β-lapachone co-treatment compared with those in the cisplatin alone group. Notably, the protective effects of β-lapachone in wild-type mice were completely abrogated in NQO1−/− mice. Moreover, β-lapachone enhanced the tumoricidal action of cisplatin in a xenograft tumor model. Thus, intracellular regulation of NAD+ levels through NQO1 activation might be a promising therapeutic target for the protection of cisplatin-induced acute kidney injury.

Recently, we demonstrated that pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6 played a critical role in cisplatin-induced cochlear injury and that flunarizine, known as a T-type Ca 2+ channel antagonist, induced a cytoprotective effect against cisplatin cytotoxicity in HEI-OC1 cells by the activation of NF-E2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) cascade through PI3K-Akt signaling but calciumindependent pathway. We report here that flunarizine markedly attenuates cisplatin-induced pro-inflammatory cytokine secretion and their messenger RNA transcription as well as cisplatin cytotoxicity through the activation of Nrf2/HO-1 and downregulation of NF-κB. In HEI-OC1 cells, overexpression of Nrf2/HO-1 by gene transfer or pharmacological approaches attenuated cisplatin-induced cytotoxicity and proinflammatory cytokine production. On the contrary, inhibition of Nrf2/HO-1 signaling by pharmacological inhibitors or specific small interfering RNAs significantly abolished the beneficial effects of flunarizine. Flunarizine also attenuated cisplatin-mediated MAPK activation and pharmacological inhibition of MAPKs, especially MEK1/ERK, blocked cisplatin-induced NF-κB activation in HEI-OC1 cells. Furthermore, WT-Nrf2 overexpression effectively blocked MAPK activation after cisplatin exposure. Finally, orally administrated Sibelium™, the trade name of flunarizine, suppressed the increase of pro-inflammatory cytokines by cisplatin in both serum and cochleas of mice, whereas it increased HO-1 expression in cochleas. These results indicate that flunarizine induces a protective effect against cisplatin ototoxicity through the downregulation of NF-κB by Nrf2/HO-1 activation and the resulting inhibition of pro-inflammatory cytokine production in vitro and in vivo.