본 연구는 이독성 약물인 젠타마이신과 시스플라틴에 의해 난청이 유발된 렛트 와우 조직, 청각 유모세포 및 특이 억제제를 사용한 실험결과들을 바탕으로 3 가지 세포사멸 기전 (아포토시스, 오토파지 그리고 네크로토시스)을 조망해 본 바, 종합적으로 젠타마이신은 주로 아포토시스와 오토파지 세포사멸
기전을 활성화시키고, 시스플라틴은 네크로토시스와 일부 오토파지를
활성화시킴으로써 서로 다른 세포사멸 기전이 복합적으로 발생되어 이독성 난청이 야기되는 것을 밝혔다.
참고 문헌
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ABSTRACT
-Cell death map in drug-induced ototoxicity:
apoptosis, autophagy, and necroptosis
Mi-Jin Choi
BK21 Plus program, Department of Biomedical Sciences, Ajou University The Graduate School of Medicine, Suwon, Republic of Korea
(Supervised by Professor Yun-Hoon Choung, MD, DDS, PhD)
Introduction: Gentamicin (GM) and Cisplatin (CDDP) show various side effects such as sensorineural hearing loss and dizziness in which the mechanisms have not been identified. The purpose of the study is to analyze drug type-induced auditory cell death mechanisms including apoptosis, autophagy, and necroptosis.
Methods: In vitro study, HEI-OC1 auditory cells were cultured and treated with various doses of GM (0, 2.5, 5, and 7.5 mM) or CDDP (0, 3.25, 7.5, and 15 μM), and then analyzed by Western blot. To analyze apoptotic effects by GM- and CDDP-induced ototoxicity, cleaved PARP, and cleaved caspase-3 bands were analyzed in Western blot.
RIP1, RIP3, and MLKL were used as the markers for necroptotic effect. For autophagy markers, Beclin1, p62, and LC3 were used. The measured of GM or CDDP-induced cell death by WST-1 assay under the pre-treatment with specific cell death inhibitors
Chloroquine (10 µM), Necrostatin-1 (10 µM), Z-VAD (0.05 µM) or combinations, respectively. In vivo study, healthy 6~8-week-old male Sprague-Dawley rats (200–350 g) were used. Auditory brainstem responses (ABRs; 8, 16 and 32 kHz) were measured at the time of pre-injection and post-injection, and hearing threshold shifts were analyzed. Each dose of GM (200 mg/kg) or CDDP (16 mg/kg) was intraperitoneally injected and then analyzed by H&E staining, TUNEL, and immunofluorescence for the morphologic study.
Results: On the 6 weeks after GM injection, hearing loss was detected, with thresholds increasing to 26.3 ± 20.0 dB, 23.8 ± 18.9 dB and 27.5 ± 21.9 dB at 8, 16 and 32 kHz, respectively. At 3 days after cisplatin injection, ABR thresholds also increased to 24.4 ± 14.2 dB, 29.7 ± 16.8 dB and 30.8 ± 18.7 dB at 8, 16 and 32 kHz, compared to the control (11.7 ± 4.0 dB). In H&E stain, we found the change of nuclear morphology, such as breakdown and shrinkage, and the decreased number of spiral ganglion cells treated with GM and CDDP. In immunofluorescence, LC3B staining was much more highly expressed than RIP1 and RIP3 in spiral ganglions and lateral ligaments in GM-induced ototoxicity.
Reversely, RIP1 and RIP3 were much higher expressed than LC3B staining in spiral ganglions and lateral ligaments under CDDP. In vitro study showed GM-induced ototoxicity, the intensity of cleaved caspase-3, Bax and LC3B under GM treatment were increased, but the intensity of pMLKL was increased at 6 h single point. In CDDP-induced ototoxicity, the intensity of pMLKL increased in a time-dependent manner. Moreover, LC3B was partially increased under CDDP treatment. Cell death was significantly inhibited by pre-treated with Z-VAD (0.05 µM), CQ (10 µM), Nec-1 (10 µM) or each combinations.
Conclusion: GM and CDDP seem to have different ototoxic mechanisms; apoptosis or autophagy in GM and necroptosis in CDDP.
Keyword: Gentamicin (GM), cisplatin (CDDP), sensorineural hearing loss, drug-induced ototoxicity, apoptosis, autophagy, necroptosis