Otos Gene Up-regulation Protects Cultured Spiral Ligament Fibrocytes Of Rats Against Cisplatin-induced Damage

Background and Objective:Application of Cisplatin, a widely used anti-cancer agent, often leads to serious ototoxicity and thus affects quality of life in patients. Increasing doses of cisplatin has been used for treatment of cancers because of their multi-drug resistance, resulting in serious ototoxicity. Studies tried to attenuate cisplatin-induced hearing loss via administration of some reagents as well as herbal medicine. However, the concentrations of the preventive drugs in cochlea were low and their efficacy on hearing loss was limited because of the presence of blood-labyrinth barrier. Hence, to find effective methods for prevention and treatment of cisplatin-induced hearing loss is required. Gene therapy is likely to be an effective and promising approach.Spiral ligament, an essential part of blood-labyrinth barrier in cochlea, has been suggested to be a key part of cochlea mediating kalium ion recycling. It is known that abrogated expression of several genes such as Cx26, Cx30, Cx31, Cx43, COCH and Pendrin being expressed by spiral ligament fibrocytes (SLFs), may ruin ionic balance in endolymph, inevitably leading to hearing loss. Of the genes found in SLFs, Otos is a novel and special gene. Evidence suggests that down-regulation of Otos causes hearing loss. Accordingly, Otos was suggested to be essential for maintenance of hearing. However, whether Otos could prevent SLFs from cisplatin-induced apoptosis remains largely unknown. In the present study, we aimed to evaluate the possible roles and underlying mechanisms by which Otos rescues SLFs from cisplatin-mediated damage.Methods:1. Expressions of Otos mRNA in rat cochleae were assessed by in situ hybridization assay.2. SLFs were primarily cultured and identified by immunofluorescence assay.3. pAdTrack-Otos was cloned by inserting the fragment of Otos into the shuttle plasmid vector pAdTrack-CMV. For obtaining recombinant adenovirus plasmid (Ad-Otos), the pAdTrack-Otos and the backbone plasmid pAdEasy-1 were homologously recombined in bacteria BJ5183, then pAdEasy-GFP-Otos was transfected into 293 cells for getting packaged infectious replicate-deficient adenoviruses.4. The constructed Ad-Otos and Ad-GFP were transfected into SLFs and the proper multiplicity of infection (MOI) and time length were evaluated.5. SLFs were transfected with Ad-Otos or Ad-GFP at a MOI of 50 for 48 h, and then the cells were divided into two groups. Each of the groups contained three subgroups, namely, SLFs-AdOtos, SLFs-AdGFP and SLFs-nontransfection. The two groups were treated with 20μM cisplatin or DMEM for 12 h, respectively. After that, the cell viability and apoptosis were assessed by MTT and TUNEL assays respectively. Otos mRNA was assessed by RT-PCR and JNK,p-JNK,ERK,p-ERK,p38,p-p38,bcl-2,bax,caspase-3 were further assessed by immunoblotting.6. The Ad-Otos transfected SLFs were divided into five groups, three of which were pretreated with 10μM SP600125, PD98059 and SB203580 respectively 1 h prior to treatment with 20μM cisplatin. The remaining 2 groups were treated with cisplatin or DMEM. After that, the cell proliferation and apoptosis were assessed by MTT and TUNEL assays and the mitochondrial pathway-related proteins were detected by immunoblotting.Results1. Otos mRNA was detected on the spiral ligaments of rats.2. The cells were successfully cultured and identified as typeⅠSLFs.3.A recombinant adenovirus with GFP report gene was constructed in 293 cells. PCR test proved the recombinant adenovirus containing the insertion of Otos. The titre of purified recombinant adenovirus was 7×109 pfu/ml.4. The proper MOI for transfection was 50.5. SLFs transfected solely with Ad-Otos resulted in increased expression of Otos mRNA, activation of MAPKs signaling pathway and inactivation of mitochondrial pathway.6. With the treatment of cisplatin, SLFs-AdOtos showed an increase in cell viability, a decrease in apoptosis rate compared with those of SLFs-AdGFP or SLFs-nontransfection, with activation of ERK pathway, comparative inactivation of JNK as well as mitochondrial pathway.7. The data suggest that JNK, ERK but not p38 might be involved in the process of Otos preventing SLFs from cisplatin damage by using signaling pathway inhibitors.ConclusionsUp-regulation of Otos might rescue cultured SLFs from cisplatin-induced cell apoptosis. In this process, blockade of JNK and activation of ERK as well as inhibition of mitochondrial pathway might be involved in its underlying mechanisms. In addition, Otos up-regulation might be a potential strategy for treatment of cisplatin-induced deafness.