Bisdemethoxycurcumin-mediated Attenuation of Apoptosis Prevents Gentamicin-induced Ototoxicity in Mouse Cochlear UB/OC-2 Cells

Abstract

Background/aim: Gentamicin has been widely prescribed since the last two decades despite its ototoxicity and nephrotoxicity. Bisdemethoxycurcumin (BDMC) is an affordable and safe curcuminoid with medicinal properties. We aimed to understand the effects of BDMC on the gentamicin-induced hair cell damage in mouse cochlear UB/OC-2 cells, in order to elucidate the therapeutic potential of BDMC against gentamicin-induced ototoxicity.

Materials and methods: We quantified the cell membrane potential and examined the regulators and cascade proteins in the intrinsic pathway of hair cell apoptosis. Mouse cochlear UB/OC-2 cells were treated with BDMC before exposure to gentamicin. The effects of BDMC on hair cell viability, mitochondrial function, and apoptosis-related proteins were examined by flow cytometry, western blot, and fluorescent staining.

Results: Our results revealed that BDMC reversed gentamicin-mediated cycle arrest at the G₂/M phase, stabilizing the mitochondrial membrane potential, decreasing cleaved caspase proteins, and successfully reversing hair cell apoptosis.

Conclusion: BDMC is a potential agent for reducing gentamicin-induced ototoxicity.

Keywords: Gentamicin; apoptosis; bisdemethoxycurcumin; mitochondrial function; ototoxicity; reactive oxygen species.

Figure 1. Effect of gentamicin and bisdemethoxycurcumin (BDMC) on cell viability and cytotoxicity in gentamicin-treated UB/OC-2 cells. Cell viability after treatment with (A) gentamicin (0–1.5 mM) and (B) BDMC (0-10 μM), for 24 h. (C) Cells were pretreated with or without 5 μM BDMC for 2 h and then incubated with or without 1.25 mM gentamicin for 24 h. The cytotoxicity was measured by the lactate dehydrogenase (LDH) release assay. Data are presented as the mean±SD of three independent experiments. *p<0.05; **p<0.01; ***p<0.001.

Figure 2. Effect of bisdemethoxycurcumin (BDMC) on cell cycle progression in gentamicin-treated UB/OC-2 cells. Cells were pretreated with or without 5 μM BDMC for 2 h, and then incubated with or without 1.25 mM gentamicin for 24 h. (A) Cell cycle distribution was measured by flow cytometry. (B) Quantitative analysis of the proportion of cells in each cycle phase. Data are presented as the mean±SD of three independent experiments. **p<0.01; ***p<0.001.

Figure 3. Effect of bisdemethoxycurcumin (BDMC) on intracellular reactive oxygen species (ROS) production and mitochondrial membrane potential in gentamicin-treated UB/OC-2 cells. (A) Cells were pretreated with or without 5 μM BDMC for 2 h, and then incubated with or without 1.25 mM gentamicin for 24 h. (A) ROS production [dichlorofluorescein diacetate (DCFDA) assay] and (B) mitochondrial permeability (JC-1 staining), respectively, and analyzed by flow cytometry. Data are presented as the mean±SD of three independent experiments. *p<0.05; **p<0.01.

Figure 4. Effect of bisdemethoxycurcumin (BDMC) on mitochondrial-related proteins in gentamicin-treated UB/OC-2 cells. Cells were pretreated with or without 5 μM BDMC for 2 h, and then incubated with or without 1.25 mM gentamicin for 24 h. Expression levels of cytochrome c in cytosolic (A) and mitochondrial fractions (B) were measured by western blot analysis. (C) Expression levels of Bcl-2 and Bax were evaluated by western blot analysis. β-actin and COΧ IV were used as loading controls. Data are presented as the mean±SD of three independent experiments. *p<0.05; **p<0.01.

Figure 5. Effect of bisdemethoxycurcumin (BDMC) on apoptosis-related proteins in gentamicin-treated UB/OC-2 cells. Cells were pretreated with or without 5 μM BDMC for 2 h, and then incubated with or without 1.25 mM gentamicin for 24 h. Expression levels of Caspase-9 and cleaved Caspase-9 (A), Caspase-3 and cleaved Caspase-3 (B), and PARP and cleaved PARP (C) were measured by western blot analysis. β-actin was used as the loading control. Data are presented as the mean±SD of three independent experiments. *p<0.05; **p<0.01; ***p<0.001.

Figure 6. Effect of bisdemethoxycurcumin (BDMC) on cell death in gentamicin-treated UB/OC-2 cells. Cells were pretreated with or without 5 μM BDMC for 2 h, and then incubated with or without 1.25 mM gentamicin for 24 h. (A) The percentage of apoptotic cells was assessed by Annexin V/propidium iodide (PI) assays using flow cytometry. Data are presented as the mean±SD of three independent experiments. *p<0.05; **p<0.01. (B) Condensed or fragmented nuclei of the apoptotic cells were observed by Hoechst 33258 staining using a fluorescence microscope (100×). Scale bar=200 μm.