Selective Delivery of a Therapeutic Gene for Treatment of Head and Neck Squamous Cell Carcinoma Using Human Neural Stem Cells

Objectives

Based on studies of the extensive tropism of neural stem cells (NSCs) toward malignant brain tumor, we hypothesized that NSCs could also target head and neck squamous cell carcinoma (HNSCC) and could be used as a cellular therapeutic delivery system.

Methods

To apply this strategy to the treatment of HNSCC, we used a human NSC line expressing cytosine deaminase (HB1.F3-CD), an enzyme that converts 5-fluorocytosine (5-FC) into 5-fluorouracil (5-FU), an anticancer agent. HB1. F3-CD in combination with 5-FC were cocultured with the HNSCC (SNU-1041) to examine the cytotoxicity on target tumor cells in vitro. For in vivo studies, an HNSCC mouse model was created by subcutaneous implantation of human HNSCC cells into athymic nude mice. HB1.F3-CD cells were injected into mice using tumoral, peritumoral, or intravenous injections, followed by systemic 5-FC administration.

Results

In vitro, the HB1.F3-CD cells significantly inhibited the growth of an HNSCC cell line in the presence of the 5-FC. Independent of the method of injection, the HB1.F3-CD cells migrated to the HNSCC tumor, causing a significant reduction in tumor volume. In comparison to 5-FU administration, HB1.F3-CD cell injection followed by 5-FC administration reduced systemic toxicity, but achieved the same level of therapeutic efficacy.

Conclusion

Transplantation of human NSCs that express the suicide enzyme cytosine deaminase combined with systemic administration of the prodrug 5-FC may be an effective regimen for the treatment of HNSCC.     

Expression of VEGF, HGF, IL-6, IL-8, MMP-9, Telomerase in Peripheral Blood of Patients with Head and Neck Squamous Cell Carcinoma

Objectives

This study investigated the telomerase expression in peripheral blood mononuclear cells (PBMCs) and the relationship between the serum level of several soluble factors such as vascular endothelial growth factor (VEGF), hepatocyte growth factor, interleukin (IL)-6, IL-8, and matrix metallopeptidase-9 and the clinicopathological features of patients with head and neck squamous cell carcinoma (HNSCC).

Methods

Peripheral blood samples were collected from 50 HNSCC patients and 15 normal controls. The telomerase activity in the PBMCs was measured by Telomere Repeat Amplification Protocols. The serum levels of the soluble factors were analyzed by enzyme-linked immunosorbent assay.

Results

The expression of telomerase in the PBMCs of HNSCC patients was significantly correlated with the N and American Joint Committee on Cancer (AJCC) stages. The serum VEGF level was significantly higher in the patients with an advanced T stage, N stage and AJCC stage. Serum VEGF was significantly related with the expression of telomerase in the PBMCs. The telomerase expression and the VEGF expression were shown to be independent factors associated with poor survival.

Conclusion

The telomerase expression in the PBMCs and the serum VEGF level of HNSCC patients were significantly correlated with the N stage, the AJCC stage and the prognosis.     

Expression Profile of Fas-Fas Ligand in Spiral Ganglion Cells During Apoptosis

Objectives

To examine the expression profile of Fas-Fas ligand (FasL) during glutamate (Glu)-induced spiral ganglion cell (SGC) apoptosis.

Methods

Cultured SGCs were treated with 10-mM, 25-mM, and 50-mM concentrations of Glu and incubated for 24 or 48 hours. The expression intensity of FasL, Fas, caspase 3, and morphology of single SGC were evaluated using immunofluorescence staining.

Results

In semiquantitative analysis of the Glu-treated SGC, FasL, and caspase 3 expression intensity were increased with concentration- and time-dependent manner. Fas expression intensity did not change with different concentration at 48 hours. In morphologic analysis of the Glu-treated SGC, number of apoptotic cells were increased with concentration- and time-dependent manner.

Conclusion

FasL was expressed in apoptotic SGCs, suggesting that the Fas-FasL signaling pathway may be involved in the Glu-induced apoptosis of dissociated SGCs.     

Expression of Bone-related Genes in Bone Marrow MSCs after Cyclic Mechanical Strain: Implications for Distraction Osteogenesis

Aim Understanding the response of mesenchymal stem cells （MSCs） to mechanical strain and their consequent gene expression patterns will broaden our knowledge of the mechanobiology of distraction osteogenesis. Methodology In this study, a single period of cyclic mechanical stretch （0.5 Hz, 2,000 με） was performed on rat bone marrow MSCs. Cellular proliferation and alkaline phosphatase （ALP） activity was examined. The mRNA expression of six bone-related genes （Ets-1, bFGF, IGF-Ⅱ, TGF-β, Cbfal and ALP） was detected using real-time quantitative RT-PCR. Results The results showed that mechanical strain can promote MSCs proliferation, increase ALP activity, and up-regulate the expression of these genes. A significant increase in Ets-1 expression was detected immediately after mechanical stimulation, but Cbfal expression became elevated later. The temporal expression pattem of ALP coincided perfectly with Cbfal. Conclusion The results of this study suggest that mechanical strain may act as a stimulator to induce differentiation of MSCs into osteoblasts, and that these bone-related genes may play different roles in the response of MSCs to mechanical stimulation.     

Neural-Induced Human Mesenchymal Stem Cells Promote Cochlear Cell Regeneration in Deaf Guinea Pigs

Objectives

In mammals, cochlear hair cell loss is irreversible and may result in a permanent sensorineural hearing loss. Secondary to this hair cell loss, a progressive loss of spiral ganglion neurons (SGNs) is presented. In this study, we have investigated the effects of neural-induced human mesenchymal stem cells (NI-hMSCs) from human bone marrow on sensory neuronal regeneration from neomycin treated deafened guinea pig cochleae.

Methods

HMSCs were isolated from the bone marrow which was obtained from the mastoid process during mastoidectomy for ear surgery. Following neural induction with basic fibroblast growth factor and forskolin, we studied the several neural marker and performed electrophysiological analysis. NI-hMSCs were transplanted into the neomycin treated deafened guinea pig cochlea. Engraftment of NI-hMSCs was evaluated immunohistologically at 8 weeks after transplantation.

Results

Following neural differentiation, hMSCs expressed high levels of neural markers, ionic channel markers, which are important in neural function, and tetrodotoxin-sensitive voltage-dependent sodium currents. After transplantation into the scala tympani of damaged cochlea, NI-hMSCs-injected animals exhibited a significant increase in the number of SGNs compared to Hanks balanced salt solution-injected animals. Transplanted NI-hMSCs were found within the perilymphatic space, the organ of Corti, along the cochlear nerve fibers, and in the spiral ganglion. Furthermore, the grafted NI-hMSCs migrated into the spiral ganglion where they expressed the neuron-specific marker, NeuN.

Conclusion

The results show the potential of NI-hMSCs to give rise to replace the lost cochlear cells in hearing loss mammals.     

Th2 Responses Elicited by Nasal Epithelial Cells Exposed to House Dust Mite Extract

Objectives

Respiratory epithelial cells are the first site of interaction of allergens with the immune system. The aim of this study was to examine the effect of epithelial cells, which were stimulated with house dust mite (HDM) extracts, on the immune response of peripheral blood mononuclear cells (PBMCs).

Methods

Primary nasal polyp epithelial cells were exposed to dermatophagoides pteronyssinus and dermatophagoides farina for 48 hr, and then the supernatant and cells were collected. After stimulation with HDM extract, the epithelial cells were co-cultured with PBMCs for 72 hr and then the supernatant was collected. We measured the interleukin (IL)-8 and granulocyte-macrophage colony stimulating factor to determine the activation of the epithelial cells. The tumor necrosis factor (TNF)-α, IL-5 and interferon-γ were measured to evaluate the interaction between the epithelial cells and the PBMCs. The mRNA expression of intercellular adhesion molecule 1 (ICAM-1) was assessed using the anti-ICAM-1 antibody.

Results

The HDM extracts activated the nasal epithelial cells and enhanced the expression of ICAM-1 mRNA and cell membrane ICAM-1. When the activated epithelial cells were co-cultured with PBMCs, the PBMCs produced lager amounts of TNF-α and IL-5. However the cytokine production was not inhibited by pretreatment with ICAM-1 antibody.

Conclusion

HDM allergens induce allergic inflammation by activating nasal epithelial cells, yet the interaction of the epitheila cells and the PBMCs may not be associated with an ICAM-1 medicated mechanism.     

Growth Inhibition and Apoptosis with H31 Metabolites from Marine Bacillus SW31 in Head and Neck Cancer Cells

Objectives

To determine whether a novel marine micro-organism with anticancer properties, H31, the metabolic product of Bacillus SW31, has anti-tumor effects on head and neck cancer, and potential for apoptotic-enhancing anti-cancer treatment of affected patients.

Methods

The cell viability and apoptosis assays were performed. Changes in the signal pathway related to apoptosis were investigated. Then, the therapeutic effects of H31 were explored in mouse xenograft model and drug toxicity of H31 was examined in zebrafish model.

Results

We identified the anticancer activity of H31, a novel metabolic product of Bacillus SW31. Bacillus SW31, a new marine micro-organism, has 70% homology with Bacillus firmus and contains potent cytotoxic bioactivity in head and neck cancer cells using MTT assay. Combined with c-JUN, p53, cytochrome C, and caspase-3, H31 induced apoptosis of KB cells, a head and neck cancer cell line. In a separate in vivo model, tumor growth in C3H/HeJ syngeneic mice was suppressed by H31. In addition, in a zebrafish model used for toxicity testing, a considerable dose of H31 did not result in embryo or neurotoxicity.

Conclusion

Growth inhibition and apoptosis were achieved both in vitro and in vivo in head and neck cancer cells after exposure to H31, a metabolite from the marine Bacillus species, without any significant toxicity effects even at considerable H31 dose concentrations.     

Antioxidant and Anti-Apoptotic Effect of Melatonin on the Vestibular Hair Cells of Rat Utricles

Objectives

Aminoglycosides are commonly used antibiotic agents, and they are known to generate free oxygen radicals within the inner ear and to cause vestibulo-cochlear toxicity and permanent damage to the sensory hair cells and neurons. Melatonin, a pineal secretory product, has the properties of being a powerful direct and indirect antioxidant. The aim of the present study was to prove the antioxidant effect of melatonin against gentamicin-induced ototoxicty.

Methods

The utricular maculae of Sprague-Dawley rats were prepared from postnatal day 2-4, and these maculae were were divided into 6 groups as follows: 1) control, 2) melatonin only, 3) gentamicin only, and 4), 5), and 6) gentamicin plus melatonin (10, 50, and 100 µM, respectively). To count the number of hair cells, 5 utricles from each group were stained with phalloidin-FITC on the 1st, 4th, and 7th days after drug administration. Reactive oxygen species (ROS) was assessed by using the fluorescent probe hydrofluorescent diacetate acetyl ester. The caspase-3 activity was also examined with using the fluorescent caspase-3 substrate and performing Western blotting.

Results

The result of this study showed that gentamicin induced the loss of utricular hair cells, and this loss of hair cells was significantly attenuated by co-administration of melatonin. Melatonin reduced ROS production and caspase-3 activation in the gentamicin treated utricular hair cells.

Conclusion

Our findings conclusively reveal that melatonin has protective effects against gentamicin-induced hair cell loss in the utricles of rat by inhibiting both ROS production and caspase-3 activity.     

Transplantation of Neural Stem Cells in Anosmic Mice

Objectives

Treating olfactory dysfunction is a challenge for physicians. One of the therapeutic options could be transplantation of stem cells. In this study, neural stem cells were transplanted into anosmic mice.

Methods

Neural stem cells were generated from the olfactory bulb of green fluorescent protein (GFP)-transgenic C57BL6 mice. Anosmia were induced by injection of intraperitoneal 3-methylindole. The neural stem cells were transplanted transnasally on the next day. The olfactory function was evaluated by a food-finding test once a week. The olfactory neuroepithelium was harvested for histologic examination and protein analysis at 4 weeks.

Results

Twenty-five percent (6/24) of the control mice that were not transplanted with neural stem cells survived at 4 weeks while 67% (8/12) of the transplanted mice survived (P=0.029). The food finding test showed that the transplanted mice resumed finding food at 3 weeks while the control mice resumed finding food at 4 weeks. GFP-positive cells were observed in the olfactory neuroepithelium of the transplanted mice. Western blotting revealed that the olfactory marker protein expression was significantly lower in the control mice than that in the transplanted mice.

Conclusion

This study demonstrated that improvement of mouse survival was achieved and recovery of olfactory function was promoted by transnasal transplantation of neural stem cells in the anosmic mouse model. These results indicate that stem cells might be one of the future modalities for treating olfactory impairment.     

Apoptosis Progression in the Hair Cells in the Organ of Corti of GJB2 Conditional Knockout Mice

Objectives

Apoptosis may play an important role in the mechanism underlying the GJB2 gene conditional knockout (cCx26) mice cochlear cell death. The objective of this study was to explore the the damage mode of the outer hair cells (OHCs) and its real time point of apoptosis and provide information to further explore the role of apoptosis in the happening of hearing loss in cCx26 mice.

Methods

Cochleae from mice at various developmental stages (P8, P12, and P21) were dissected out and first used to be observed under the scanning electron microscope (SEM). Basilar membranes from mice at P8, P14, P18, and P21 were stained by fluorescein isothiocyanate-conjugated phalloidin and propidium iodide (PI) and examined under confocal microscope.

Results

The loss of OHCs of cCx26 knockout mice was first set between P12 and P21 under SEM. Whole mount phalloidin and PI staining revealed that obvious apoptotic appearance of the OHCs surface morphology was observed at P18.

Conclusion

Typical apoptotic morphology was found in the OHCs in the organ of Corti of the cCx26 mice at P18. This may provide information to further study the role of apoptosis in the occurrence of hearing loss of cCx26 mice.     

Protective Effect of Metformin on Gentamicin-Induced Vestibulotoxicity in Rat Primary Cell Culture

Objectives

One of the antidiabetic drugs, metformin, have shown that it prevented oxidative stress-induced death in several cell types through a mechanism involving the opening of the permeability transition pore and cytochrome c release. Thus, it is possible that the antioxidative effect of metformin can also serve as protection against gentamicin-induced cytotoxicity related to reactive oxygen species (ROS). The aim of this study was to examine the protective effect of metformin on gentamicin-induced vestibulotoxicity in primary cell culture derived from rat utricle.

Methods

For vestibular primary cell culture, rat utricles were dissected and incubated. Gentamicin-induced cytotoxicity was measured in both the auditory and vestibular cells. To examine the effects of metformin on gentamicin-induced cytotoxicity in the primary cell culture, the cells were pretreated with metformin at a concentration of 1 mM for 24 hours, and then exposed to 2.5 mM gentamicin for 48 hours. The intracellular ROS level was measured using a fluorescent dye, and also measured using a FACScan flow cytometer. Intracellular calcium levels in the vestibular cells were measured with calcium imaging using Fura-2 AM.

Results

Vestibular cells were more sensitive to gentamicin-induced cytotoxicity than auditory hair cells. Metformin protects against gentamicin-induced cytotoxicity in vestibular cells. Metformin significantly reduced a gentamicin-induced increase in ROS, and also reduced an increase in intracellular calcium concentrations in gentamicin-induced cytotoxicity.

Conclusion

Metformin significantly reduced a gentamicin-induced increase in ROS, stabilized the intracellular calcium concentration, and inhibited gentamicin-induced apoptosis. Thus, Metformin showed protective effect on gentamicin-induced cytotoxicity in vestibular primary cell culture.     

CXCL12/SDF-1α Activates NF-κB and Promotes Oral Cancer Invasion through the Carma3/Bcl10/Maltl Complex

Aim To determine how SDF-1α/CXCR4 activates nuclear factor-kappa B （NF-κB） and promotes oral squamous cell carcinoma （OSCC） invasion.Methodology A lentivirus-based knockdown approach was utilized to deplete gene expression. NF-κB activation was evaluated by Western blot analysis and electrophoretic mobility shift （EMSA）. Results We show that the activation of NF-κB by CXCR4 occurs through the Carma3/Bcl10/Maltl （CBM） complex in OSCC. We found that loss of components of the CBM complex in HNSCC can inhibit SDF-1α induced phosphorylation and degradation of IκBα, while TNFα induced IKK activation remains unchanged. Further, we identified a role for novel and atypical, but not classical, PKCs in activating IKK through CXCR4. Importantly, inhibition of the CBM complex leads to a significant decrease in SDF-1α mediated invasion of OSCC. Conclusion The CBM complex plays a critical role in CXCR4-induced NF-κB activation in OSCC. Targeting molecular components of the NF-κB signaling pathway may provide an important therapeutic opportunity in controlling the progression and metastasis of OSCC mediated by SDF-1α.     

Expression of EGFR and Microvessel Density in Middle Ear Cholesteatoma

Objectives

Cholesteatoma destructs bony tissue by the interactions between hyperproliferative epithelial cells and subepithelial inflammatory cells. The aim of this study was to evaluate the expression of epidermal growth factor receptor (EGFR) and microvessel density (MVD) in middle ear cholesteatoma tissue in an effort to determine the relationship between expression of EGFR and neovascularization.

Methods

We evaluated the expression of EGFR and MVD by immunohistochemical staining for CD31 and Factor VIII in 32 cholesteatoma tissue samples and 7 normal postauricular skin samples. We also analyzed the correlation between EGFR expression and MVD.

Results

The expression of EGFR was higher in cholesteatoma than in postauricular skin, but the difference was not statistically significant. EGFR was more highly expressed in the suprabasal layer than in the basal layer. Using CD31 and Factor VIII, we analyzed the MVD and found that it was significantly higher in cholesteatoma than in postauricular skin, and significantly correlated with the expression of EGFR.

Conclusion

Our results suggest that overexpression of EGFR and neovascularization are correlated with the growth of cholesteatoma.     

Effect of Epigallocatechin-3-Gallate on PMA-Induced MUC5B Expression in Human Airway Epithelial Cells

Objectives

Among the inflammatory mediators, phorbol 12-myristate 13-acetate (PMA) is associated with the regulation of MUC5B expression in the airway epithelial cells. Epigallocatechin-3-gallate (EGCG) is the major component of green tea extract. The biological activity of EGCG includes reduction of cholesterol and antioxidant activity, as well as anti-inflammatory effect. However, the precise action mechanism of anti-inflammatory effect of EGCG in the airway epithelial cells has not been fully defined. This study investigates the effect and the brief signaling pathway of EGCG on PMA-induced MUC5B expression in the airway epithelial cells.

Methods

In NCI-H292 airway epithelial cells, the effect and signaling pathway of EGCG on MUC5B expression were investigated using real-time polymerase chain reaction analysis, enzyme immunoassay, immunohistochemical analysis, gelatin zymography assay, and immunoblot analysis.

Results

In NCI-H292 airway epithelial cells, PMA induced MUC5B expression, phosphorylation of p38 mitogen-activated protein kinase (MAPK), and matrix metalloproteinase-9 (MMP-9) expression and protein activity. EGCG significantly decreased PMA-induced MUC5B expression, phosphorylation of p38 MAPK, and MMP-9 expression and protein activity. SB203580 (p38 MAPK inhibitor) significantly decreased PMA-induced MMP-9 expression. In addition, SB203580 and MMP-9 I (MMP-9 inhibitor) significantly decreased PMA-induced MUC5B expression.

Conclusion

These results suggest that EGCG down-regulates PMA-induced MUC5B expression through the p38 MAPK dependent MMP-9 signaling pathway in human airway epithelial cells.     

Apoptotic Pattern of Cochlear Outer Hair Cells and Frequency-specific Hearing Threshold Shift in Noise-exposed BALB/c Mice

Objectives

Apoptosis of outer hair cell (OHC) can be identified through nuclear staining by specific nuclear changes. The change of filamentous actin (F-actin) is also involved in early cell death process. The study was designed to investigate OHC death along the whole length of the organ of Corti.

Methods

BALB/c hybrid mice were used in this study. The noise group was exposed to white noise of 120 dB SPL for 3 hr per day for 3 consecutive days. The tone burst auditory brainstem response (ABR) test was conducted and cochleas from each group were obtained for the immunostaining of FITC phalloidin for F-actin and propidium iodide (PI) for nuclei.

Results

ABR threshold of the noise group significantly increased after noise exposure (P<0.001). No threshold shift was found in the control group. Threshold shift of the noise group constantly increased from 4 to 16 kHz, but threshold shifts at 16 kHz and 32 kHz were similar. Patterns of OHC staining were subclassified as FITC+PI- cells, FITC+ PI+ cells, FITC-PI+ cells and missing cells. Proportion of normal live OHCs (FITC+PI-) rapidly decreased from the apex to the base. In the basal turn, FITC-PI+ cells and vacancy OHC (missing cells) were observed easily. Apoptotic and missing cells were most abundant at 60% of the whole length of the Corti organ.

Conclusion

We could subclassify morphologic changes in OHC death after noise exposure. Quantitative changes in OHCs along the whole Corti organ showed a plateau pattern similar to that of a frequency-specific threshold shift.     

Establishment and Characterization of an In Vitro Model for Cholesteatoma

Objectives

Experimental models are of importance to study the pathogenesis of middle ear cholesteatoma, however, they were not established until now. We aimed to develop in vitro model of middle ear cholesteatoma using primary keratinocytes and fibroblasts isolated from cholesteatoma tissue. HaCaT cell line was used as a "skin equivalent" and to compare the grade of homogeneity between cholesteatoma keratinocytes and HaCaT cells.

Methods

Primary keratinocytes were isolated from cholesteatoma tissue, co-cultured with preliminary prepared feeder layer from cholesteatoma fibroblasts and subsequently air-exposed. The protein profile of cholesteatoma keratinocytes and HaCaT cells was evaluated by means of immunoblot using monoclonal antibody against cytokeratin (CK) 13 and 16. Tissue localization of CK 13 and 16 was accomplished with immunohistochemistry.

Results

Different protein profile and stronger expression of CK 13 and 16 were demonstrated in cholesteatoma keratinocytes in comparison with HaCaT cells. Bigger stratification was observed in the 3D-in vitro systems when both cholesteatoma keratinocytes and HaCaT cells were respectively co-cultured with fibroblasts in comparison with the corresponding control groups without fibroblasts.

Conclusion

3D-model demonstrates the significance of intercellular interaction between components of cholesteatoma tissue.     

Risk Factors and Survival Outcomes for Patients With Anastomotic Leakage After Surgery for Head and Neck Squamous Cell Carcinoma

Objectives

This study evaluated the risk factors for anastomotic leakage (AL) and survival outcomes in patients with head and neck squamous cell carcinoma (HNSCC).

Methods

Patients with HNSCC who underwent surgery carrying potential AL from 2003 through 2009 were included in this study. Univariate and multivariate analyses were performed and patient survival was calculated by the Kaplan-Meier method.

Results

Of 232 eligible patients, 25 (10.8%) developed AL. Univariate analyses revealed that primary tumor site, salvage surgery, perineural invasion, radiotherapy, chemotherapy, and blood transfusion were significantly associated with the occurrence of AL (P<0.05). Independent risk factors for AL were salvage surgery and blood transfusion (P<0.01). On univariate analysis, AL was significantly associated with overall (OS) and disease-free survivals (DFS; P<0.05) but not with decreased locoregional control (LRC) rate (P=0.07). The 5-year DFS rate was significantly different between the non-leakage and leakage groups (70.9% vs. 27.7%, P<0.001). Multivariate analysis showed, however, that AL was not an independent variable of LRC, DFS, or OS (P>0.1).

Conclusion

Patients who received salvage surgery and blood transfusion may require careful surveillance for development of AL, which has a tendency toward decreased survival.     

Temperature Enhances Activation and Inactivation Kinetics of Potassium Currents in Inner Hair Cells Isolated from Guinea-Pig Cochlea

Objectives

Until recently, most patch-clamp recordings in inner hair cells (IHCs) have been performed at room temperature. The results acquired at room temperature should be corrected if they are to be related to in vivo findings. However, the temperature dependency to ion channels in IHCs is unknown. The aim of this study was to investigate the effect of temperature on the potassium currents in IHCs.

Methods

IHCs were isolated from a mature guinea-pig cochlea and potassium currents were recorded at room temperature (around 25℃) and physiological temperatures (35℃-37℃).

Results

IHCs showed outwardly rectifying currents in response to depolarizing voltage pulses, with only a slight inward current when hyperpolarized. The amplitude of both outward and inward currents demonstrated no temperature dependency, however, activation and inactivation rates were faster at 36℃ than at room temperature. Half-time for activation was shorter at 36℃ than at room temperature at membrane potentials of -10, +10, +20, +30, and +40 mV. Q₁₀ for the activation rate was 1.83. The inactivation time constant in outward tetraethylammonium-sensitive potassium currents was much smaller at 36℃ than at room temperature between the membrane potentials of -20 and +60 mV. Q₁₀ for the inactivation time constant was 3.19.

Conclusion

The results of this study suggest that the amplitude of potassium currents in IHCs showed no temperature dependence either in outward or inward-going currents, however, activation and inactivation accelerated at physiological temperatures.     

Protective Effect of Minocycline Against Cisplatin-induced Ototoxicity

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.