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Hepatocyte growth factor mimetic protects lateral line hair cells from aminoglycoside exposure.

Uribe PM, Kawas LH, Harding JW, Coffin AB - Front Cell Neurosci (2015)

Bottom Line: We found that a Dihexa concentration of 1 μM confers optimal protection from acute treatment with either ototoxin.Pretreatment with Dihexa does not affect the amount of fluorescently tagged gentamicin that enters hair cells, indicating that Dihexa's protection is likely mediated by intracellular events and not by inhibiting aminoglycoside entry.Our data suggest that Dihexa confers protection of hair cells through an HGF-mediated mechanism and that Dihexa holds clinical potential for mitigating chemical ototoxicity.

View Article: PubMed Central - PubMed

Affiliation: Department of Integrative Physiology and Neuroscience, Washington State University Pullman, WA, USA.

ABSTRACT
Loss of sensory hair cells from exposure to certain licit drugs (e.g., aminoglycoside antibiotics, platinum-based chemotherapy agents) can result in permanent hearing loss. Here we ask if allosteric activation of the hepatocyte growth factor (HGF) cascade via Dihexa, a small molecule drug candidate, can protect hair cells from aminoglycoside toxicity. Unlike native HGF, Dihexa is chemically stable and blood-brain barrier permeable. As a synthetic HGF mimetic, it forms a functional ligand by dimerizing with endogenous HGF to activate the HGF receptor and downstream signaling cascades. To evaluate Dihexa as a potential hair cell protectant, we used the larval zebrafish lateral line, which possesses hair cells that are homologous to mammalian inner ear hair cells and show similar responses to toxins. A dose-response relationship for Dihexa protection was established using two ototoxins, neomycin and gentamicin. We found that a Dihexa concentration of 1 μM confers optimal protection from acute treatment with either ototoxin. Pretreatment with Dihexa does not affect the amount of fluorescently tagged gentamicin that enters hair cells, indicating that Dihexa's protection is likely mediated by intracellular events and not by inhibiting aminoglycoside entry. Dihexa-mediated protection is attenuated by co-treatment with the HGF antagonist 6-AH, further evidence that HGF activation is a component of the observed protection. Additionally, Dihexa's robust protection is partially attenuated by co-treatment with inhibitors of the downstream HGF targets Akt, TOR and MEK. Addition of an amino group to the N-terminal of Dihexa also attenuates the protective response, suggesting that even small substitutions greatly alter the specificity of Dihexa for its target. Our data suggest that Dihexa confers protection of hair cells through an HGF-mediated mechanism and that Dihexa holds clinical potential for mitigating chemical ototoxicity.

No MeSH data available.


Related in: MedlinePlus

Dihexa-mediated protection is inhibited by the HGF antagonist 6-AH. (A) Treatment with 1 μM 6-AH does not alter lateral line hair cell survival in response to neomycin (Two-way ANOVA; 6-AH: F(1,72) = 3.255 p = 0.0754). (B) Treatment with 1 μM Dihexa decreases neomycin-induced hair cell death over multiple concentrations of neomycin. Co-treatment with 6-AH completely attenuates Dihexa-mediated protection (Two-way ANOVA; Dihexa: F(2,76) = 76.30 p < 0.001). There is a significant difference (p < 0.001) when comparing 1 μM Dihexa vs. 1 μM Dihexa plus 1 μM 6-AH at 50, 100, and 200 μM neomycin treatment groups, p < 0.05 at 400 μM neomycin. N = 6–9 animals per treatment, error bars represent ± s.e.m.
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Figure 5: Dihexa-mediated protection is inhibited by the HGF antagonist 6-AH. (A) Treatment with 1 μM 6-AH does not alter lateral line hair cell survival in response to neomycin (Two-way ANOVA; 6-AH: F(1,72) = 3.255 p = 0.0754). (B) Treatment with 1 μM Dihexa decreases neomycin-induced hair cell death over multiple concentrations of neomycin. Co-treatment with 6-AH completely attenuates Dihexa-mediated protection (Two-way ANOVA; Dihexa: F(2,76) = 76.30 p < 0.001). There is a significant difference (p < 0.001) when comparing 1 μM Dihexa vs. 1 μM Dihexa plus 1 μM 6-AH at 50, 100, and 200 μM neomycin treatment groups, p < 0.05 at 400 μM neomycin. N = 6–9 animals per treatment, error bars represent ± s.e.m.

Mentions: We used the pharmacological inhibitor 6-AH to investigate if the observed protection conferred by Dihexa is actually mediated by HGF/c-Met signaling. 6-AH blocks HGF dimerization and thereby inhibits subsequent c-Met activation (Kawas et al., 2012). Treatment with 6-AH alone does not alter hair cell survival in response to neomycin (Figure 5A). Co-treatment of larvae with 6-AH and Dihexa completely attenuates the hair cell protection conferred by Dihexa treatment (Figure 5B). This observation implicates the requirement of an active HGF/c-Met signal in Dihexa otoprotection.


Hepatocyte growth factor mimetic protects lateral line hair cells from aminoglycoside exposure.

Uribe PM, Kawas LH, Harding JW, Coffin AB - Front Cell Neurosci (2015)

Dihexa-mediated protection is inhibited by the HGF antagonist 6-AH. (A) Treatment with 1 μM 6-AH does not alter lateral line hair cell survival in response to neomycin (Two-way ANOVA; 6-AH: F(1,72) = 3.255 p = 0.0754). (B) Treatment with 1 μM Dihexa decreases neomycin-induced hair cell death over multiple concentrations of neomycin. Co-treatment with 6-AH completely attenuates Dihexa-mediated protection (Two-way ANOVA; Dihexa: F(2,76) = 76.30 p < 0.001). There is a significant difference (p < 0.001) when comparing 1 μM Dihexa vs. 1 μM Dihexa plus 1 μM 6-AH at 50, 100, and 200 μM neomycin treatment groups, p < 0.05 at 400 μM neomycin. N = 6–9 animals per treatment, error bars represent ± s.e.m.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4309183&req=5

Figure 5: Dihexa-mediated protection is inhibited by the HGF antagonist 6-AH. (A) Treatment with 1 μM 6-AH does not alter lateral line hair cell survival in response to neomycin (Two-way ANOVA; 6-AH: F(1,72) = 3.255 p = 0.0754). (B) Treatment with 1 μM Dihexa decreases neomycin-induced hair cell death over multiple concentrations of neomycin. Co-treatment with 6-AH completely attenuates Dihexa-mediated protection (Two-way ANOVA; Dihexa: F(2,76) = 76.30 p < 0.001). There is a significant difference (p < 0.001) when comparing 1 μM Dihexa vs. 1 μM Dihexa plus 1 μM 6-AH at 50, 100, and 200 μM neomycin treatment groups, p < 0.05 at 400 μM neomycin. N = 6–9 animals per treatment, error bars represent ± s.e.m.
Mentions: We used the pharmacological inhibitor 6-AH to investigate if the observed protection conferred by Dihexa is actually mediated by HGF/c-Met signaling. 6-AH blocks HGF dimerization and thereby inhibits subsequent c-Met activation (Kawas et al., 2012). Treatment with 6-AH alone does not alter hair cell survival in response to neomycin (Figure 5A). Co-treatment of larvae with 6-AH and Dihexa completely attenuates the hair cell protection conferred by Dihexa treatment (Figure 5B). This observation implicates the requirement of an active HGF/c-Met signal in Dihexa otoprotection.

Bottom Line: We found that a Dihexa concentration of 1 μM confers optimal protection from acute treatment with either ototoxin.Pretreatment with Dihexa does not affect the amount of fluorescently tagged gentamicin that enters hair cells, indicating that Dihexa's protection is likely mediated by intracellular events and not by inhibiting aminoglycoside entry.Our data suggest that Dihexa confers protection of hair cells through an HGF-mediated mechanism and that Dihexa holds clinical potential for mitigating chemical ototoxicity.

View Article: PubMed Central - PubMed

Affiliation: Department of Integrative Physiology and Neuroscience, Washington State University Pullman, WA, USA.

ABSTRACT
Loss of sensory hair cells from exposure to certain licit drugs (e.g., aminoglycoside antibiotics, platinum-based chemotherapy agents) can result in permanent hearing loss. Here we ask if allosteric activation of the hepatocyte growth factor (HGF) cascade via Dihexa, a small molecule drug candidate, can protect hair cells from aminoglycoside toxicity. Unlike native HGF, Dihexa is chemically stable and blood-brain barrier permeable. As a synthetic HGF mimetic, it forms a functional ligand by dimerizing with endogenous HGF to activate the HGF receptor and downstream signaling cascades. To evaluate Dihexa as a potential hair cell protectant, we used the larval zebrafish lateral line, which possesses hair cells that are homologous to mammalian inner ear hair cells and show similar responses to toxins. A dose-response relationship for Dihexa protection was established using two ototoxins, neomycin and gentamicin. We found that a Dihexa concentration of 1 μM confers optimal protection from acute treatment with either ototoxin. Pretreatment with Dihexa does not affect the amount of fluorescently tagged gentamicin that enters hair cells, indicating that Dihexa's protection is likely mediated by intracellular events and not by inhibiting aminoglycoside entry. Dihexa-mediated protection is attenuated by co-treatment with the HGF antagonist 6-AH, further evidence that HGF activation is a component of the observed protection. Additionally, Dihexa's robust protection is partially attenuated by co-treatment with inhibitors of the downstream HGF targets Akt, TOR and MEK. Addition of an amino group to the N-terminal of Dihexa also attenuates the protective response, suggesting that even small substitutions greatly alter the specificity of Dihexa for its target. Our data suggest that Dihexa confers protection of hair cells through an HGF-mediated mechanism and that Dihexa holds clinical potential for mitigating chemical ototoxicity.

No MeSH data available.


Related in: MedlinePlus