Protective Effects of Glucose-Related Protein 78 and 94 on Cisplatin-Mediated Ototoxicity

Cisplatin is a widely used chemotherapeutic drug for treating various solid tumors. Ototoxicity is a major dose-limiting side effect of cisplatin, which causes progressive and irreversible sensorineural hearing loss. Here, we examined the protective effects of glucose-related protein (GRP) 78 and 94, also identified as endoplasmic reticulum (ER) chaperone proteins, on cisplatin-induced ototoxicity. Treating murine auditory cells (HEI-OC1) with 25 μM cisplatin for 24 h increased cell death resulting from excessive intracellular reactive oxygen species (ROS) accumulation and caspase-involved apoptotic signaling pathway activation with subsequent DNA fragmentation. GRP78 and GRP94 expression was increased in cells treated with 3 nM thapsigargin or 0.1 μg/mL tunicamycin for 24 h, referred to as mild ER stress condition. This condition, prior to cisplatin exposure, attenuated cisplatin-induced ototoxicity. The involvement of GRP78 and GRP94 induction was demonstrated by the knockdown of GRP78 or GRP94 expression using small interfering RNAs, which abolished the protective effect of mild ER stress condition on cisplatin-induced cytotoxicity. These results indicated that GRP78 and GRP94 induction plays a protective role in remediating cisplatin-ototoxicity

Antioxidant Therapy against Oxidative Damage of the Inner Ear: Protection and Preconditioning

Oxidative stress is an important mechanism underlying cellular damage of the inner ear, resulting in hearing loss. In order to prevent hearing loss, several types of antioxidants have been investigated; several experiments have shown their ability to effectively prevent noise-induced hearing loss, age-related hearing loss, and ototoxicity in animal models. Exogenous antioxidants has been used as single therapeutic agents or in combination. Antioxidant therapy is generally administered before the production of reactive oxygen species. However, post-exposure treatment could also be effective. Preconditioning refers to the phenomenon of pre-inducing a preventative pathway by subtle stimuli that do not cause permanent damage in the inner ear. This renders the inner ear more resistant to actual stimuli that cause permanent hearing damage. The preconditioning mechanism is also related to the induction of antioxidant enzymes. In this review, we discuss the mechanisms underlying antioxidant-associated therapeutic effects and preconditioning in the inner ear

Induction of Redox-Active Gene Expression by CoCl2 Ameliorates Oxidative Stress-Mediated Injury of Murine Auditory Cells

Free radicals formed in the inner ear in response to high-intensity noise, are regarded as detrimental factors for noise-induced hearing loss (NIHL). We reported previously that intraperitoneal injection of cobalt chloride attenuated the loss of sensory hair cells and NIHL in mice. The present study was designed to understand the preconditioning effect of CoCl2 on oxidative stress-mediated cytotoxicity. Treatment of auditory cells with CoCl2 promoted cell proliferation, with increases in the expressions of two redox-active transcription factors (hypoxia-inducible factor 1α, HIF-1α, nuclear factor erythroid 2-related factor 2; Nrf-2) and an antioxidant enzyme (peroxiredoxin 6, Prdx6). Hydrogen peroxide treatment resulted in the induction of cell death and reduction of these protein expressions, reversed by pretreatment with CoCl2. Knockdown of HIF-1α or Nrf-2 attenuated the preconditioning effect of CoCl2. Luciferase reporter analysis with a Prdx6 promoter revealed transactivation of Prdx6 expression by HIF-1α and Nrf-2. The intense immunoreactivities of HIF-1α, Nrf-2, and Prdx6 in the organ of Corti (OC), spiral ganglion cells (SGC), and stria vascularis (SV) of the cochlea in CoCl2-injected mice suggested CoCl2-induced activation of HIF-1α, Nrf-2, and Prdx6 in vivo. Therefore, we revealed that the protective effect of CoCl2 is achieved through distinctive signaling mechanisms involving HIF-1α, Nrf-2, and Prdx6

Protective effect of DEX on cell viability, ROS generation, and expression of apoptosis-related proteins in TNF-α-treated cells.

Cells were pretreated with 10 nM DEX for 6 h, followed by treatment with 10 ng/ml TNF-α for a further 24 h. (A) The effect of DEX pretreatment on cell viability reduced by TNF-α. Data in the graph are expressed as means ± SE of four independent experiments. *,# P #; TNF-α only versus DEX plus TNF-α. (B) Measurement of intracellular ROS accumulation using CM-H2DCFDA. Values are expressed as means ± SE for three independent experiments. *,# P #; TNF-α only versus DEX plus TNF-α. (C) Representatives immunoblot of apoptosis-related protein expression. Each protein expression was normalized by that of β-actin. The values in a graph are expressed as fold changes relative to the untreated control and expressed as means ± SE of three independent experiments. *,# P P P #; TNF-α only versus DEX plus TNF-α.</p

Protective effects of DEX on TNF-α-induced cochlear hair cells loss in cochlear explants.

(A) The Representative confocal images of IHCs and OHCs stained by myosin 7a in middle-turn cochlear explants (a; control, b; 10 nM DEX, c; 20 ng/ml TNF-α, d; 10 nM DEX pretreatment then 20 ng/ml TNF-α). Scale bars = 20 mm, Original magnification = 400 ×. (B,C) The graph represents the number of myosin 7a positive IHCs or OHCs per 160 mm in the middle-turn cochlear explants, expressed as a mean ± SE (n = 3 different explants per group); *,# P ,### P ,***; compared with untreated control, #,###; TNF-α only versus DEX plus TNF-α.</p

Attenuation of TNF-α-induced apoptosis by DEX pretreatment.

Cells were pretreated with DEX for 6 h before exposure to TNF-α for 24 h. (A) Representative images of Annexin V (green) and PI (red) staining in HEI-OC1 cells. Scale bar = 100 μm, Original magnification = 100 ×. (B) Values in the graphs are presented the percentage of apoptotic cells. The data in graphs are expressed as means ± SE (n = 3). *,# P P P ,**,***; compared with untreated control, #; TNF-α only versus DEX plus TNF-α.</p

Effects of TNF-α on the cochlear hair cells in middle-turn cochlear explants.

(A) The representative confocal images show the middle-turn cochlear explants treated with a culture medium alone (control) or a medium containing different concentrations of TNF-α (10, 20, and 40 ng/ml). After treatment, explants were fixed, permeabilized, and stained with a polyclonal myosin 7a antibody as a hair cell marker. Scale bars = 20 mm, Original magnification = 400 ×. (B, C) Quantification of myosin 7a positive Inner hair cells (IHCs) and Outer hair cells (OHCs) per 160 mm in the middle-turn cochlear explants, respectively. Data are expressed as mean ± SE of the number of IHCs or OHCs (n = 3 different explants per group); * P P ,***; compared with untreated control.</p

Effects of DEX and TNF-α on intracellular ROS accumulation and expression of apoptosis-related proteins.

Cells were treated with 10 nM DEX for 6 h or 10 ng/ml TNF-α for 24 h. (A) Measurement of ROS generation using DCF fluorescence intensities. Values are expressed as the means ± SE for three independent experiments, expressed as a fold change of untreated control value. * P P < 0.05 compared with untreated control.</p

Fig 1 -

Effects of TNF-α and dexamethasone (DEX) on cell viability (A) Dose-dependent cell viability of HEI-OC1 cells exposed to TNF-α. Cells were treated with various concentrations of TNF-α (0–20 ng/ml) for 24 h and viability was measured using a CCK-8 assay. (B) Cell viability of HEI-OC1 cells at various concentrations of DEX. Cells were treated with DEX (0–20 nM) for 6 h or 24 h and viability was measured using a CCK-8 assay. All values are expressed as the means ± SE for four independent experiments, expressed as a percentage of untreated control value. * P < 0.05, ** P < 0.01, *** P < 0.001 compared with untreated control.</p