NAC attenuates cochlear degeneration in S1pr2^−/− mice. (A) S1pr2^−/− mice were treated with vehicle or NAC (1 g/kg/day, p.o.) between 1 and 5 weeks of age, then evaluated by ASR. A small cohort of S1pr^+/− littermates were similarly evaluated at 5 weeks as a reference for normal ASR response. (B) Representative appearance of the organs of Corti in the basal turn of the cochlea at 6 weeks of age. S1pr2^+/− mice uniformly present with intact organs of Corti containing the normal complement of three outer hair cells (arrow). S1pr2^−/− mice were characterized by near-complete degeneration (asterisks). S1pr2^−/− mice treated with NAC demonstrated decreased degeneration, although many organs of Corti were characterized by structural abnormalities (arrowhead). (C) Quantitation of the organ of Corti degeneration at 6 weeks of age. Degeneration indicates complete loss of the organ of Corti. (n = 12, * p < 0.05, ** p < 0.01, *** p < 0.001.).

CYM-5478 prevents cisplatin-mediated ABR abnormalities. (A) Rats were treated with cisplatin for 3 weeks, with or without co-administration of CYM-5478, before hearing acuity was evaluated by ABR. Although all groups demonstrated similar response thresholds of ~35 dB, the cisplatin-only group exhibited increased waveform latency at all click intensities (arrow). Stimulus onset is at ~7 ms for all groups, including the time taken for sound to reach the ear. (B) Quantification of waveform V latency of 90 dB stimuli shown in (A). (n = 3–4, * p < 0.05, ** p < 0.01, ns = not significant.).

CYM-5478 attenuates cisplatin-mediated degeneration of zebrafish hair cells. (A) CYM-5478-mediated activation of the zebrafish homolog of S1P₂ (zS1P₂) was evaluated by the receptor-specific TGFα-shedding assay. Human S1P₂ (hS1P₂) was used as a reference. (B) Representative images of zebrafish larvae treated with cisplatin in the presence of NAC (1 mM) or CYM-5478 (20 μM). Viable neuromasts were labeled with YO-PRO-1. (C) Quantification of YO-PRO-1-positive neuromasts. (*** p < 0.001, n = 4).

CYM-5478 attenuates apoptosis and oxidative stress in neural cells. (A) C6 glioma cells were treated with cisplatin (20 μM) for 24 h in the presence of increasing concentrations of CYM-5478, then evaluated for caspase 3/7 activity. (n = 6.) (B) cDNA prepared from CLU-188 mouse hypothalamic cells and 4T1 mouse mammary carcinoma cells was amplified with gene-specific primers. S1P₂ is the most abundant S1P receptor present in both cell lines. (C) CLU-188 cells and 4T1 cells were treated with cisplatin (20 μM) overnight in the presence or absence of CYM-5478, then evaluated for ROS using the CellROX assay. (n = 5, * p < 0.05, ** p < 0.01).

CYM-5478 promotes anti-apoptotic pathways in neural but not breast cancer cells. Cisplatin (20 µM) and CYM5478 (20 µM) either alone or in combination were treated for 24 h, after which western blot analysis was performed. CYM-5478 reverses cisplatin-induced suppression of anti-apoptotic phospho-STAT3 and Bcl-xL while inhibiting expression of pro-apoptotic Bax in CLU-188 mouse neural cells. CYM5478 had no effect on cisplatin-induced suppression of phospho-STAT3 or Bcl-xL, and did not suppress Bax in 4T1 mouse breast cancer cells.