A putative role of p53 pathway against impulse noise induced damage as demonstrated by protection with pifithrin-alpha and a Src inhibitor.

Exposure to high-level noise leads to oxidative stress and triggers apoptosis of the hair cells. This study examined whether p53, a tumor suppressor protein, is activated in the cochlea following impulse noise exposure. Inhibition of p53 with pifithrin alpha, a specific p53 inhibitor, or KX1-004, a Src-protein tyrosine kinase inhibitor, was tested to determine if p53 inhibition could reduce noise-induced hearing loss and cochlear damage. Chinchillas were pre-treated with a local administration of pifithrin alpha or KX1-004 and exposed to impulse noise. The chinchillas were assessed for threshold shift at 1 and 24h after the noise. At 4 or 24h post noise, the cochleae were removed and organs of Corti were examined to assess the damage to the cells and upregulation of p53 by the noise. Apoptosis was evident in both outer hair cells and supporting cells. Phospho-p53 (Ser 15) was upregulated 4h and 24h after the noise. KX1-004 and pifithrin alpha both decreased threshold shift and the number of missing outer hair cells. These results indicate that p53 is involved in the early stages of noise-induced cell death and inhibition of this signaling pathway is a potential protective strategy against noise-induced hearing loss.

Regulation of PSA secretion and survival signaling by calcium-independent phopholipase A(2)beta in prostate cancer cells.

BACKGROUND: Serum prostate specific antigen (PSA) levels in prostate cancer patients serve as a useful biomarker for diagnosing and monitoring prostate cancer. Recently, secreted PSA has been characterized as an autocrine survival factor through activation of Akt and induction of AR. In the normal prostate, PSA is secreted in the lumen of prostatic ducts to lyse proteins in the seminal coagulum. METHODS: However, the mechanism for constitutive PSA secretion from benign prostate and its transport across the prostate-blood barrier into serum are unknown. Regulation of peptide secretion by iPLA(2)-beta has been reported in non-prostatic tissue and in prostate tissue iPLA(2)-beta is reported to be under androgen regulation. We investigated whether iPLA(2) plays a role for in PSA secretion by comparing iPLA(2) activity and expression in normal prostate epithelial RWPE-1 cells and in LNCaP prostate cancer cells. Expression of the two active iPLA(2)-beta mRNA splice variants, LH-iPLA(2) and SH-iPLA(2), were increased and the inhibitory ankyrin-iPLA(2) isoform was markedly reduced in LNCaP cells as compared to normal prostate epithelial RWPE-1 cells. RESULTS: These changes are consistent with a higher enzymatic activity in LNCaP cells. The iPLA(2)-beta-specific inhibitor BEL inhibited PSA secretion and induced apoptosis in LNCaP cells. iPLA(2) knockdown using SiRNA inhibited PSA secretion, downregulated AR and induced apoptosis. Exogenous PSA suppressed BEL-induced apoptosis and neutralizing anti-PSA antibody blocked the survival effect of PSA. CONCLUSIONS: These data indicate that iPLA(2)-beta participates in regulating PSA secretion and supports the concept that secreted PSA provides an autocrine survival function in LNCaP cells.

Linear quantitation of Abeta aggregation using Thioflavin T: reduction in fibril formation by colostrinin.

Thioflavin T (ThT) fluorescence is a commonly used method to monitor Abeta protein fibril formation. This method is particularly attractive since ThT fluoresces only when bound to fibrils, the reaction is completed within 1min and ThT does not interfere with aggregation of Abeta fibrils. One of the drawbacks of this method is the lack of a strict quantitative relationship between ThT fluorescence and fibril content. It was observed that, when the same gram molecular weight of Abeta (1-40) is dissolved into varying amounts of base then placed into a constant volume of aqueous buffer, a non-linear fluorescent response is obtained. By maintaining a strict relationship between Abeta content and the volume of base, this anomalous result can be alleviated and a linear dose response curve is obtained at much lower Abeta concentrations than is typically observed. In addition, differences in Abeta batch to batch preparations are alleviated. It was previously reported that colostrinin (CLN), a proline-rich peptide derived from colostrum, reduces fibril content and protects neuroblastoma cells against Abeta peptide-induced toxicity. The newly developed ThT fluorescence protocol was used to quantify Abeta fibril content after treatment with CLN. We also demonstrate that CLN, can solubilize Abeta fibrils in a dose and time-dependent fashion.

RETRACTED: Prevention of hearing loss from noise exposure using Src inhibitors.

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Extremely rapid induction of outer hair cell apoptosis in the chinchilla cochlea following exposure to impulse noise.

We have reported the presence of OHC apoptosis and necrosis in the organ of Corti following exposure to intense noise. The current study was designed to investigate the rapidity and the initial pattern of outer hair cell (OHC) death induced by exposure to impulse noise. Chinchillas were exposed to 75 pairs of impulse noise at 155 dB peak sound pressure level presented over a time period of 75 s. At 5 or 30 min after the noise exposure, the cochleae were examined for morphological and biological indicators of apoptosis and necrosis. In the cochleae collected within 5 min after the 75-s noise exposure, there were clear signs of nuclear condensation and cell body shrinkage, suggesting the presence of OHC apoptosis. Apoptotic OHCs were further detected by positive staining of TUNEL and caspase-3 assays. In contrast to the rapid development of nuclear condensation, appearance of nuclear swelling, a necrotic phenotype, appeared at 30 min after the noise exposure. The results of the study demonstrate that induction of OHC apoptosis after the noise exposure is an extremely rapid process.

Protective effect of colostrinin on neuroblastoma cell survival is due to reduced aggregation of beta-amyloid.

Colostrinin (CLN), a mixture of proline-rich polypeptides, has shown a stabilizing effect on cognitive function in Alzheimer's patients measured by the Alzheimer's disease Assessment Scale-cognitive (ADAS-cog) and in Instrumental Activities of Daily Living (ILDL) in recently conducted clinical trials. The aim of this study was to elucidate a possible mode of action of CLN in the treatment of Alzheimer's disease. Here, we report that CLN prevents the aggregation of beta-amyloid peptide Abeta (1-40) in vitro. The impact of CLN on the fibril formation was monitored by optical and electron microscopy. The electron micrographs illustrate that, at 25 microM, Abeta (1-40) peptides formed fibrils after 24-48 h of incubation. The presence of 0.25 microM CLN completely abolished the fibril formation. Abeta (1-40) peptides grow into dense fibers when examined at the 20th day. In the presence of CLN, however, the fibrils are much shorter and less dense. Addition of CLN as late as the 17th day can still dissolves the preformed fibrils. These observations were compared to the effect of CLN on the neurotoxic activity of beta-amyloid peptides in the cell culture model (SHSY-5Y). The beta-amyloid peptides were pre-incubated with CLN at various times and used to treat SHSY-5Y neuroblastoma cells for up to 4 days. The cytotoxic effect was monitored by trypan blue exclusion. We demonstrated that 24-48 h treatment was the onset of toxicity of 10-50 microM of beta-amyloid peptides. Pre-incubation of 0.0025-0.25muM of CLN with 25 microM of beta-amyloid peptides leads to near-complete abolition of cytotoxicity. Low doses of CLN (2.5 nM) can attain cytotoxic protection levels similar to those of highest doses (0.25 microM). Thus, the time course for the appearance of beta-amyloid fibrils coincides with that for cytotoxicity, and that the reduction of fibrils of beta-amyloid peptides by CLN is concomitant with the reduction of the cytotoxic effects of beta-amyloid on SHSY-5Y neuroblastoma cells. Our studies suggest that the neuroprotective effects exerted by CLN are related to the reduction of beta-amyloid fibrils.

Paraquat-induced hair cell damage and protection with the superoxide dismutase mimetic m40403.

Some forms of ototoxicity appear to be mediated primarily by the superoxide radical; however, the exact role the superoxide radical plays in cochlear damage is not well understood because most ototoxic drugs produce multiple reactive oxygen species. To characterize the role of the superoxide radical in cochlear damage and the protective effect of compounds that inactivate superoxide, we treated mouse cochlear organotypic cultures for 24 h with paraquat, an herbicide that produces high levels of superoxide. M40403, a highly specific, nonpeptidyl mimetic of superoxide dismutase, was added to some cultures to inactivate the superoxide radical generated by paraquat. The number of outer hair cells (OHC) and inner hair cells (IHC) systematically decreased with increasing concentration of paraquat (0.01-10 mM). M40403 (10 muM) significantly increased OHC and IHC survival in cultures treated with 0.01-1.0 mM of paraquat. These results suggest that excess production of superoxide radical is a sufficient condition for hair cell loss.

Quantitative analysis of apoptotic and necrotic outer hair cells after exposure to different levels of continuous noise.

We have reported that by 2 days after noise exposure the size of cochlear lesion was expanding by outer hair cells (OHCs) dying either by apoptosis or necrosis. The current study was designed to compare the prevalence of the two cell death pathways as a function of time after exposure to noises of different levels. Chinchillas were exposed to a narrow band noise at either 104 or 108 dB SPL for 1 h. At three time points (1, 4 and 30 days) after the noise exposure, the numbers of missing, apoptotic and necrotic OHCs in the cochleas were identified and documented with a combination of TUNEL, caspase-3 and propidium iodide labeling. The subjects exposed to the 108-dB noise showed significantly more apoptotic OHCs than necrotic OHCs in the cochleas examined at days 1 and 4 after the noise exposure. By day 30, apoptotic and necrotic pathologies continued, although in small quantity, with no significant difference in quantity between two types of cell death. The subjects exposed to the 104-dB noise showed a significant difference in the numbers of apoptotic and necrotic OHCs at day 1 after the noise exposure, whereas the difference became statistically insignificant at day 4 and day 30 after the noise exposure. The results of the study indicate that the early expansion of cochlear lesion is attributed primarily to apoptosis, whereas the later stage of lesion expansion is likely the result of an equal contribution from apoptosis and necrosis.

The caspase pathway in noise-induced apoptosis of the chinchilla cochlea.

We previously reported that intense noise exposure causes outer hair cell (OHC) death primarily through apoptosis. Here we investigated the intracellular signal pathways associated with apoptotic OHC death. Chinchillas were exposed to a 4 kHz narrowband noise at 110 dB SPL for 1 h. After the noise exposure, the cochleas were examined for the activity of each of three caspases, including caspase-3, -8, or -9 with carboxyfluorescein-labeled fluoromethyl ketone (FMK)-peptide inhibitors. The cochleas were further examined for cytochrome c release from mitochondria by immunohistology and for DNA degradation by the TUNEL method. The results showed that the noise exposure triggered activation of caspase-3, an important mediator of apoptosis. The noise exposure also caused the activation of caspase-8 and caspase-9, each of which is associated with a distinct signaling pathway that leads to activation of caspase-3. Caspase activation occurred only in the apoptotic OHCs and not in the necrotic OHCs. These results indicate that multiple signaling pathways leading to caspase-3 activation take place simultaneously in the apoptotic OHCs. In addition to caspase activation, noise exposure caused the release of cytochrome c from mitochondria, resulting in a punctate fluorescence in the cytosol. In contrast to activation of caspases, the release of cytochrome c took place in both apoptotic and necrotic OHCs. Moreover, the release of cytochrome c in a subpopulation of OHCs took place early in the cell death process, prior to any outward signs of necrosis or apoptosis. These data suggest that in this subpopulation there exists a common step that is shared by cell death pathways before entering either necrosis or apoptosis. Lastly, use of the TUNEL assay in combination with PI labeling provides a more accurate discrimination between apoptosis and necrosis.

Noise-induced hearing loss in chinchillas pre-treated with glutathione monoethylester and R-PIA.

The protective effects of glutathione monoethylester (GEE) and GEE in combination with R-N6-phenylisopropyladenosine (R-PIA) were evaluated in the chinchilla when exposed to impulse (145 dB pSPL) or continuous (105 dB SPL, 4 kHz OB) noise. Six groups of 10 chinchillas were used as subjects. Before exposure to noise, the subjects were anesthetized, a 30 microl drop of drug was placed on the round window (GEE [50, 100, 150 mM], GEE 50 mM and R-PIA). Forty minutes later the subject was exposed to either impulse or continuous noise. The 50 mM treatment provided significant protection from impulse noise, but not from continuous noise exposure. The combination provided significant protection from both the continuous and impulse noise. In a separate set of experiments, glutathione (GSH) levels were measured in the perilymph. All the drug treatments elevated GSH levels. The results are discussed in terms of antioxidant treatments as a prophylactic measure against noise-induced hearing loss.

F-actin cleavage in apoptotic outer hair cells in chinchilla cochleas exposed to intense noise.

Apoptosis is an active cell death pathway involved in a variety of pathological conditions, including noise-induced outer hair cell (OHC) death. During this process, the cytoskeletal proteins have been found to be either damaged and/or enzymatically disassembled in several cell types, leading to formation of apoptotic manifestations. This study was designed to examine the cleavage of filamentous actin (F-actin), an important cytoskeletal protein, in the cochlear OHCs after noise exposure. Chinchillas were exposed to a 4 kHz narrow band noise at 106 dB SPL for 1 h and cochleas were either collected immediately or 3 h after the noise exposure. The organs of Corti were double-stained using FITC-labeled phalloidin for F-actin and propidium iodide for OHC nuclei. The effect of noise on F-actin and nuclei was examined by confocal microscopy. The result showed that the fluorescence associated with F-actin was decreased in the OHCs possessing condensed nuclei, but remained unchanged in the OHCs with swollen nuclei. The change in F-actin labeling occurred coordinately with the changes in nuclear morphology of apoptotic cells and was prevented by administration of caspase-3 inhibitor (Z-DEVD-FMK). The results of this study indicate that F-actin cleavage is an important early cellular event in apoptotic development in OHCs following exposure to traumatic noise.

Involvement of apoptosis in progression of cochlear lesion following exposure to intense noise.

It has been known for some time that noise-induced outer hair cell (OHC) death in the cochlea continues well after the termination of a noise exposure. However, the underlying mechanisms leading to the expansion of a cochlear lesion are not fully understood. Here we report involvement of the apoptotic pathway in the progression of OHC death in the chinchilla cochlea following exposure to a 4 kHz narrow band noise at 110 dB SPL for 1 h. Morphological examination of OHC nuclei revealed nuclear condensation and fragmentation, typical morphological features of apoptosis. OHC apoptosis developed asymmetrically toward the apical and basal parts of the cochleas following the noise exposure. Two days after the noise exposure, there was still active OHC pathology with condensed and fragmented nuclei in the basal part of the cochleas. Detection of caspase-3 activation, an intracellular marker for apoptosis, showed a spatial agreement between the apoptotic nuclei and activated caspase-3. These results clearly implicate the apoptotic pathway in the post-exposure progression of OHC demise.