Hydroxytyrosol enhances cisplatin-induced ototoxicity: Possible relation to the alteration in the activity of JNK and AIF pathways.

Hydroxytyrosol (HT), a polyphenol widely contained as an ester in olive fruits and olive leaves, exhibits a broad spectrum of effectiveness. The present study was designed to investigate the effect of HT alone as well as in the combination with cisplatin on the House Ear Institute-Organ of Corti 1 cells (HEI-OC1) and C57BL/6 cochlear hair cells in vitro. The cell viability was measured by cell counting kit-8 (CCK8) assay. The levels of reactive oxygen species were evaluated by Dichloro-dihydro-fluorescein diacetate (DCFH-DA) staining. The expression of phosphorylated Jun N-terminal kinase (p-JNK) and cleaved-caspase 3 was assessed by Western blotting. The apoptosis was detected by terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) staining. The distribution of apoptosis inducing factor (AIF) was determined by immunofluorescent staining. HT alleviated the levels of reactive oxygen species in both untreated state and after cisplatin stimulus. However, HT at concentration of 100 μM decreased the cell viability of HEI-OC1 from 100 ± 17.38% in control group to 50.17 ± 1.89% and increased the expression of p-JNK and c-caspase 3 from 0.62 ± 0.10, 0.20 ± 0.050 in the control group to 1.24 ± 0.18, 0.85 ± 0.18 in the group treated with 30 μM cisplatin, as well as to 1.64 ± 0.14, 1.44 ± 0.12 in the group with 30 μM cisplatin +100 μM HT, respectively. Meanwhile, HT triggered AIF transferring to nuclei and, also, led to cochlear HCs arranging disorderly and missing. Moreover, HT elevated the expression of p-JNK and c-caspase 3 from 1.00 ± 0.27, 1.00 ± 0.26 in the control group to 2.23 ± 0.24, 22.87 ± 3.80 in the group with 30 μM cisplatin, and to 2.75 ± 0.23, 31.56 ± 3.86 in the group with 30 μM cisplatin+100 μM HT correspondingly. Taken together, data from this work reveal that HT itself possesses toxic effect on HCs mainly thorough AIF-dependent apoptosis, while, it aggravates the ototoxicity-caused by cisplatin via both JNK and AIF pathways related apoptosis. Findings from this work offer clear evidence that that HT might not be recommended to utilize for preventing cisplatin-induced ototoxicity.