Clin Exp Otorhinolaryngol.  2011 Jun;4(2):77-82.

Protective Effect of Minocycline Against Cisplatin-induced Ototoxicity

• Lee CK
• Shin JI
• Cho YS

Abstract

OBJECTIVES
Cisplatin, a widely used chemotherapeutic agent, has serious side effects, including nephrotoxicity and ototoxicity. Minocycline is a semisynthetic second-generation tetracycline that exerts anti-inflammatory and neuroprotective effects. The purpose of this study was to elucidate the protective effect of minocycline against cisplatin-induced ototoxicity in the auditory hair cell.
METHODS
The House Ear Institute-Organ of Corti 1 (HEI-OC1) cell line and guinea pigs were used for in vitro and in vivo experiments. Cells were exposed to cisplatin with or without pre-treatment with minocycline. Cell survival was analyzed using MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide). Whole-cell lysates were collected and immunoblotted with antibodies against Bcl-2, p-c-Jun, active caspase-3, cleaved poly (ADP-ribose) polymerase (PARP), and apoptosis-inducing factor (AIF). The guinea pigs received intraperitoneal injections of cisplatin alone or following minocycline pretreatment. The auditory brainstem response was tested and the cochleae were harvested and evaluated using scanning electron microscopy.
RESULTS
Survival significantly increased in cells pretreated with minocycline compared with cells exposed to cisplatin alone. Cisplatin treatment increased the expression of active caspase 3, p-c Jun, PARP, and AIF, and pretreatment with minocycline attenuated this response. In animal study, the threshold shift by cisplatin injection in the auditory brainstem response was less pronounced in animals pretreated with minocycline. Scanning electron microscopy revealed more severe damage to the outer hair cells at the basal and middle turns than the apical turn.
CONCLUSION
Minocycline partially protects against cisplatin-induced ototoxicity via both caspase-dependent and independent apoptosis pathways.

Keyword

Minocycline; Hair cell; Apoptosis; Caspase

MeSH Terms

Animals
Antibodies
Apoptosis
Apoptosis Inducing Factor
Caspase 3
Cell Line
Cell Survival
Cisplatin
Cochlea
Ear
Electrons
Evoked Potentials, Auditory, Brain Stem
Guinea Pigs
Hair
Injections, Intraperitoneal
Microscopy, Electron, Scanning
Minocycline
Neuroprotective Agents
Tetracycline
Antibodies
Apoptosis Inducing Factor
Caspase 3
Cisplatin
Minocycline
Neuroprotective Agents
Tetracycline

Figure

• 

  Fig. 1 (A) Cell survival curve of House Ear Institute-Organ of Corti 1 (HEI-OC1) cells cultured with minocycline. Minocyline (mino) is cytotoxic at high, but not at low, concentrations. (B) Cell survival curve of HEI-OC1 cells cultured with cisplatin alone and with cisplatin (cis) after minocycline pretreatment. More cells survived in the 10 µM minocycline pretreatment condition compared with the cisplatin alone condition.

• 

  Fig. 2 Cells pretreated with 10 µM minocycline and cultured in 4 µg/mL or 8 µg/mL cisplatin were analyzed using the Western blotting technique with antibodies targeting Bcl-2, p-JUN, cleaved caspase-3, cleaved polymerase (PARP), and AIF. Bcl-2 expression was elevated after the pretreatment with minocycline. Minocycline pretreatment decreased cisplatin-induced cleaved caspase 3 activity at the 4 µg/mL, but not the 8 µg/mL dose. The expression of p-JUN, cleaved PARP, and apoptosis-inducing factor (AIF) increased by cisplatin treatment, and suppressed by minocycline pretreatment.

• 

  Fig. 3 The distribution (A) and mean amount (B) of hearing threshold shift in each group. The shift was greater in group 2 (white circle) than it was in group 1 (black circle). The threshold shift in the 16 Hz tone burst significantly different between the two groups. The threshold shift was less than 10 dB in two animals pretreated with minocycline.

• 

  Fig. 4 Scanning electron micrographs of the basal turn of the organ of Corti. (A) In a hearing-preserved animal pretreated with minocycline followed by cisplatin treatment, the outer hair cells are well preserved with minimal loss. (B) In an animal treated with cisplatin alone, catastrophic destruction occurred in the outer hair cells with fusion of cilia and bulla formation. The inner hair cells are relatively well preserved compared with the outer hair cells in both groups.

• 

  Fig. 5 The cisplatin-induced apoptosis pathway in House Ear Institute-Organ of Corti 1 (HEI-OC1) cells and the proposed mechanism mediating the protective effect of minocycline. The black arrow (→) indicates activation, and the blocked arrow head (→|) indicates inhibition. Our data show that minocycline activates Bcl-2 and decreases cleaved caspase-3, poly (ADP-ribose) polymerase (PARP), apoptosis-inducing factor (AIF) activity. These findings and those of previous studies suggest that minocycline activates Bcl-2 and inhibits downstream caspase-3 in the caspase-dependent pathway, and suppresses PARP-1 and downstream AIF in the caspase-independent pathway.

Reference

1. Hamers FP, Klis SF, Gispen WH, Smoorenburg GF. Application of a neuroprotective ACTH(4-9) analog to affect cisplatin ototoxicity: an electrocochleographic study in guinea pigs. Eur Arch Otorhinolaryngol. 1994; 251(1):23–29. PMID: 8179863.

2. Ravi R, Somani SM, Rybak LP. Mechanism of cisplatin ototoxicity: antioxidant system. Pharmacol Toxicol. 1995; 6. 76(6):386–394. PMID: 7479581.

3. Davis CA, Nick HS, Agarwal A. Manganese superoxide dismutase attenuates Cisplatin-induced renal injury: importance of superoxide. J Am Soc Nephrol. 2001; 12. 12(12):2683–2690. PMID: 11729237.

4. Boulikas T, Vougiouka M. Cisplatin and platinum drugs at the molecular level: review. Oncol Rep. 2003; Nov-Dec. 10(6):1663–1682. PMID: 14534679.

5. Domercq M, Matute C. Neuroprotection by tetracyclines. Trends Pharmacol Sci. 2004; 12. 25(12):609–612. PMID: 15530637.

6. Chen M, Ona VO, Li M, Ferrante RJ, Fink KB, Zhu S, et al. Minocycline inhibits caspase-1 and caspase-3 expression and delays mortality in a transgenic mouse model of Huntington disease. Nat Med. 2000; 7. 6(7):797–801. PMID: 10888929.

7. Wang J, Wei Q, Wang CY, Hill WD, Hess DC, Dong Z. Minocycline up-regulates Bcl-2 and protects against cell death in mitochondria. J Biol Chem. 2004; 5. 07. 279(19):19948–19954. PMID: 15004018.

8. Corbacella E, Lanzoni I, Ding D, Previati M, Salvi R. Minocycline attenuates gentamicin induced hair cell loss in neonatal cochlear cultures. Hear Res. 2004; 11. 197(1-2):11–18. PMID: 15504599.

9. Zong WX, Ditsworth D, Bauer DE, Wang ZQ, Thompson CB. Alkylating DNA damage stimulates a regulated form of necrotic cell death. Genes Dev. 2004; 6. 01. 18(11):1272–1282. PMID: 15145826.

10. Xu Y, Huang S, Liu ZG, Han J. Poly(ADP-ribose) polymerase-1 signaling to mitochondria in necrotic cell death requires RIP1/TRAF2-mediated JNK1 activation. J Biol Chem. 2006; 3. 31. 281(13):8788–8795. PMID: 16446354.

11. Yrjanheikki J, Keinanen R, Pellikka M, Hokfelt T, Koistinaho J. Tetracyclines inhibit microglial activation and are neuroprotective in global brain ischemia. Proc Natl Acad Sci U S A. 1998; 12. 22. 95(26):15769–15774. PMID: 9861045.

12. Du Y, Ma Z, Lin S, Dodel RC, Gao F, Bales KR, et al. Minocycline prevents nigrostriatal dopaminergic neurodegeneration in the MPTP model of Parkinson's disease. Proc Natl Acad Sci U S A. 2001; 12. 04. 98(25):14669–14674. PMID: 11724929.

13. Tikka T, Fiebich BL, Goldsteins G, Keinanen R, Koistinaho J. Minocycline, a tetracycline derivative, is neuroprotective against excitotoxicity by inhibiting activation and proliferation of microglia. J Neurosci. 2001; 4. 15. 21(8):2580–2588. PMID: 11306611.

14. Brundula V, Rewcastle NB, Metz LM, Bernard CC, Yong VW. Targeting leukocyte MMPs and transmigration: minocycline as a potential therapy for multiple sclerosis. Brain. 2002; 6. 125(Pt 6):1297–1308. PMID: 12023318.

15. Zhu S, Stavrovskaya IG, Drozda M, Kim BY, Ona V, Li M, et al. Minocycline inhibits cytochrome c release and delays progression of amyotrophic lateral sclerosis in mice. Nature. 2002; 5. 02. 417(6884):74–78. PMID: 11986668.

16. Wang X, Zhu S, Drozda M, Zhang W, Stavrovskaya IG, Cattaneo E, et al. Minocycline inhibits caspase-independent and -dependent mitochondrial cell death pathways in models of Huntington's disease. Proc Natl Acad Sci U S A. 2003; 9. 02. 100(18):10483–10487. PMID: 12930891.

17. Alano CC, Kauppinen TM, Valls AV, Swanson RA. Minocycline inhibits poly(ADP-ribose) polymerase-1 at nanomolar concentrations. Proc Natl Acad Sci U S A. 2006; 6. 20. 103(25):9685–9690. PMID: 16769901.

18. Alano CC, Swanson RA. Players in the PARP-1 cell-death pathway: JNK1 joins the cast. Trends Biochem Sci. 2006; 6. 31(6):309–311. PMID: 16679020.

19. Cheng PW, Liu SH, Hsu CJ, Lin-Shiau SY. Correlation of increased activities of Na+, K+-ATPase and Ca2+-ATPase with the reversal of cisplatin ototoxicity induced by D-methionine in guinea pigs. Hear Res. 2005; 7. 205(1-2):102–109. PMID: 15953519.

20. Li G, Liu W, Frenz D. Cisplatin ototoxicity to the rat inner ear: a role for HMG1 and iNOS. Neurotoxicology. 2006; 1. 27(1):22–30. PMID: 16125245.