Protective effect of metformin against cisplatin-induced ototoxicity in an auditory cell line

Abstract

Metformin, an antidiabetic drug with potent anticancer activity, is known to prevent oxidative stress-induced cell death in several cell types through a mechanism dependent on the mitochondria. In the present study, we investigated the influence of metformin on cisplatin ototoxicity in an auditory cell line. Cell viability was determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (Sigma, St. Louis, MO, USA) cell proliferation assay. Oxidative stress and apoptosis were assessed by flow cytometry analysis, Hoechst 33258 staining, reactive oxygen species (ROS) measurement, and western blotting. Intracellular calcium concentration changes were detected using calcium imaging. Pretreatment with 1 mM metformin prior to the application of 20 μM cisplatin significantly decreased the frequency of late apoptosis in HEI-OC1 cells and also significantly attenuated the cisplatin-induced increase in ROS. In addition, metformin inhibited the activation of caspase-3 and levels of poly-ADP-ribose polymerase (PARP). Pretreatment with metformin prevented the cisplatin-induced elevation in intracellular calcium concentrations. We propose that metformin protects against cisplatin-induced ototoxicity by inhibiting the increase in intracellular calcium levels, preventing apoptosis, and limiting ROS production.

FIG. 1

Cytotoxic effect of cisplatin on HEI-OC1 cells. Viable cells were counted using the MTT assay. This figure is expressed as a percentage of the untreated control cells in a time- and dose-dependent manner. Cells were treated with 10, 20, or 40 μM of cisplatin for 24 and 48 h. When cells were treated with 10, 20, and 40 μM cisplatin for 24 h, the cell viability was 97.2 (±1.2), 83.6 (±3.7), and 55.3 (±5.2) %, respectively. In addition, the cell viability for 10, 20, and 40 μM cisplatin for 48 h were 80.2(±6.1), 48.0 (±6.99), and 27.6 (±2.9) %, respectively. Twenty micomoles of cisplatin for 48 h was selected for further experiments (P < 0.001, for comparison between the control and 20 μM).

FIG. 2

Calcium imaging. In the control group, there was no change in intracellular calcium concentration until the application of ionomycin (P = 0.889, A). There was no change in intracellular calcium concentration when metformin (1 mM) was applied (B) at the 30th cycle (90 s; P = 0.262), but when cisplatin (6 mM) was applied, there was a prompt elevation of intracellular calcium concentrations (P < 0.001, C). However, when cells were pretreated with metformin (1 mM) for 24 h, intracellular calcium concentrations did not change significantly after the addition of cisplatin at the 30th cycle (6 mM; P = 0.877, D). Orange arrow, application of the agents at the 30th cycle. Purple arrow, application of the ionomycin around 310th cycle.

FIG. 3

MTT assay. When cultured cells were exposed to 20 μM cisplatin and 1 mM metformin, cell viability was 53.2 (±5.4) and 95.5 (±3.3) %, respectively. But when the cells were exposed to 20 μM cisplatin after pretreatment with 1 mM metformin for 24 h, cell viability was 69.3 (±11.1)% which was significantly higher than in the cells treated with cisplatin alone (P = 0.015).

FIG. 4

Flow cytometry analysis. There were no significant differences (P = 0.779) in the percentage of late apoptosis between controls (1.4 %) and metformin-added group (0.6 %). When 20 μM cisplatin was applied (C), the proportion of cells experiencing late apoptosis increased compared with the control (59.2 %, P = 0.012). The proportion of late apoptosis decreased when 1 mM of metformin was pretreated before the application of cisplatin (20.8 %), and the differences between (C) and (D) were statistically significant (P = 0.017).

FIG. 5

Measurement of intracellular ROS production and Hoechst 33258 staining. Apoptosis was evaluated based on the appearance of condensed and fragmented nuclei in Hoechst 33258 staining (A–E). As compared with control cells and those treated with metformin (A, B), cells exposed to 20 μM cisplatin for 48 h displayed nuclei that were condensed and fragmented (C). Group (C) also exhibited reduced cell density compared with groups (A) and (B). However, when the cells were pretreated with metformin (D), they were less condensed and less fragmented. Intracellular ROS production was measured with DCFH-DA (F–J). Cisplatin increased ROS levels (H); metformin attenuated this increase in ROS levels (I). H₂O₂ was applied for the positive control (E, J).

FIG. 6

Measurement of ROS production. ROS levels increased 1.8 ± 0.1-fold after exposure to 20 μM cisplatin. ROS levels increased 1.5 ± 0.1-fold in cells pretreated with metformin. The differences between these two values were statistically significant (P = 0.008). H₂O₂ was applied as the positive control (1.9 ± 0.6).

FIG. 7

Measurement of caspase-3 activity. Cisplatin increased caspase-3 activity (2.5 ± 0.7-fold over the normal control). The pretreatment of HEI-OC1 cells with metformin, however, significantly decreased caspase-3 activity (1.4 ± 0.2-fold over the normal control), as compared with cells treated with cisplatin alone (P = 0.001).

FIG. 8

Western blot analysis of PARP. Cisplatin increased PARP cleavage, which facilitates cellular disassembly and thereby identifies cells undergoing apoptosis. Cisplatin increased cleaved PARP by 3.9 ± 1.9-fold over the normal control. However, metformin pretreatment reduced the cleaved PARP by 2.3 ± 0.4-fold over the control. The difference between two groups were statistically significant (P = 0.009).