Protective Effects of Glucose-Related Protein 78 and 94 on Cisplatin-Mediated Ototoxicity.
ABSTRACT: Cisplatin is a widely used chemotherapeutic drug for treating various solid tumors. Ototoxicity is a major dose-limiting side effect of cisplatin, which causes progressive and irreversible sensorineural hearing loss. Here, we examined the protective effects of glucose-related protein (GRP) 78 and 94, also identified as endoplasmic reticulum (ER) chaperone proteins, on cisplatin-induced ototoxicity. Treating murine auditory cells (HEI-OC1) with 25 ?M cisplatin for 24 h increased cell death resulting from excessive intracellular reactive oxygen species (ROS) accumulation and caspase-involved apoptotic signaling pathway activation with subsequent DNA fragmentation. GRP78 and GRP94 expression was increased in cells treated with 3 nM thapsigargin or 0.1 ?g/mL tunicamycin for 24 h, referred to as mild ER stress condition. This condition, prior to cisplatin exposure, attenuated cisplatin-induced ototoxicity. The involvement of GRP78 and GRP94 induction was demonstrated by the knockdown of GRP78 or GRP94 expression using small interfering RNAs, which abolished the protective effect of mild ER stress condition on cisplatin-induced cytotoxicity. These results indicated that GRP78 and GRP94 induction plays a protective role in remediating cisplatin-ototoxicity.
Project description:Ototoxicity is a serious health problem that greatly affects millions of people worldwide. This condition is caused by the entry of aminoglycosides into auditory hair cells, subsequently inducing reactive oxygen species (ROS) production and accumulation. Several strategies have been adopted to overcome irreversible ROS-induced hair cell loss in mammals. In recent years, icariin, a major active component of the traditional herb Epimedium, has been widely studied and revealed to have antioxidant, anti-inflammatory, and anti-apoptotic properties. In this study, we found that icariin pretreatment improved the survival rate of gentamicin-treated House Ear Institute-Organ of Corti 1 (HEI-OC1) cells and cochlear explants. Icariin remarkably suppressed HEI-OC1 cell apoptosis and inhibited ROS production in cells. Notably, icariin upregulated PGC-1? (SIRT3 promoter) and SIRT3 expression in HEI-OC1 cells. In addition, SIRT3 inhibition significantly attenuated the anti-apoptotic effect of icariin. We also found that icariin can increase AMPK phosphorylation. Further studies showed that inhibition of SIRT3 activity had no significant effect on AMPK phosphorylation. Furthermore, the AMPK inhibitor compound C significantly suppressed SIRT3 expression, meaning that AMPK, as an upstream molecule, regulates SIRT3 expression. Meanwhile, inhibition of AMPK activity significantly reduced the protective effect of icariin on gentamicin ototoxicity. Based on these results, icariin exerts its protective effect on gentamicin-induced ototoxicity via activation of the AMPK-SIRT3 signaling pathway, thus providing a new strategy for treating ototoxicity caused by aminoglycoside antibiotics.
Project description:BACKGROUND:Ototoxicity is one of the major side effects of platinum-based chemotherapy, especially cisplatin therapy. To date, no FDA approved agents to alleviate or prevent this ototoxicity are available. However, ototoxicity is generally believed to be produced by excessive generation of reactive oxygen species (ROS) in the inner ear, thus leading to the development of various antioxidants, which act as otoprotective agents. Astaxanthin (ATX) is an interesting candidate in the development of new therapies for preventing and treating oxidative stress-related pathologies, owing to its unique antioxidant capacity. METHODS AND RESULTS:In this study, we aimed to evaluate the potential antioxidant properties of ATX in the inner ear by using the HEI-OC1 cell line, zebrafish, and guinea pigs. Because ATX has poor solubility and cannot pass through round window membranes (RWM), we established lipid-polymer hybrid nanoparticles (LPN) for loading ATX. The LPN enabled ATX to penetrate RWM and maintain concentrations in the perilymph in the inner ear for 24 h after a single injection. ATX-LPN were found to have favorable biocompatibility and to strongly affect cisplatin-induced generation of ROS, on the basis of DCFHDA staining in HEI-OC1 cells. JC-1 and MitoTracker Green staining suggested that ATX-LPN successfully reversed the decrease in mitochondrial membrane potential induced by cisplatin in vitro and rescued cells from early stages of apoptosis, as demonstrated by FACS stained with Annexin V-FITC/PI. Moreover, ATX-LPN successfully attenuated OHC losses in cultured organ of Corti and animal models (zebrafish and guinea pigs) in vivo. In investigating the protective mechanism of ATX-LPN, we found that ATX-LPN decreased the expression of pro-apoptotic proteins (caspase 3/9 and cytochrome-c) and increased expression of the anti-apoptotic protein Bcl-2. In addition, the activation of JNK induced by CDDP was up-regulated and then decreased after the administration of ATX-LPN, while P38 stayed unchanged. CONCLUSIONS:To best of our knowledge, this is first study concluded that ATX-LPN as a new therapeutic agent for the prevention of cisplatin-induced ototoxicity.
Project description:Cisplatin-induced ototoxicity is one of the major adverse effects in cisplatin chemotherapy, and hearing protective approaches are unavailable in clinical practice. Recent work unveiled a critical role of autophagy in cell survival in various types of hearing loss. Since the excessive activation of autophagy can contribute to apoptotic cell death, whether the activation of autophagy increases or decreases the rate of cell death in CDDP ototoxicity is still being debated. In this study, we showed that CDDP induced activation of autophagy in the auditory cell HEI-OC1 at the early stage. We then used rapamycin, an autophagy activator, to increase the autophagy activity, and found that the cell death significantly decreased after CDDP injury. In contrast, treatment with the autophagy inhibitor 3-methyladenine (3-MA) significantly increased cell death. In accordance with in vitro results, rapamycin alleviated CDDP-induced death of hair cells in zebrafish lateral line and cochlear hair cells in mice. Notably, we found that CDDP-induced increase of Sirtuin 1 (SIRT1) in the HEI-OC1 cells modulated the autophagy function. The specific SIRT1 activator SRT1720 could successfully protect against CDDP-induced cell loss in HEI-OC1 cells, zebrafish lateral line, and mice cochlea. These findings suggest that SIRT1 and autophagy activation can be suggested as potential therapeutic strategies for the treatment of CDDP-induced ototoxicity.
Project description:Cisplatin, a small platinum-containing molecule, is a widely used, highly effective anticancer drug. However, severe side effects have been found in cancer patients treated with cisplatin, including nephrotoxicity, neurotoxicity, and ototoxicity. These cisplatin-induced side effects can have a major impact on patient quality of life, including social development problems in pediatric patients that develop hearing loss. Previous studies have suggested that the major cause of cisplatin-induced ototoxicity is abnormal accumulation of reactive oxygen species (ROS) and oxidative stress. Alpha-lipoic acid (ALA), one of the most effective antioxidants, is known to be involved in the cellular antioxidant system and may have a protective effect on cisplatin-induced ototoxicity. However, the therapeutic effect of ALA on damaged hearing function and its detailed mechanism of action are not fully understood. This study focused on determining whether ALA has a potential as a protective and/or therapeutic agent for cisplatin-induced ototoxicity. Histological and physiological analyses were performed using cisplatin-treated mouse cochlea and HEI-OC1 culture cells in pre- and post-treatment with ALA in vitro and in vivo. We found that ALA contributes to protecting mitochondrial function by preventing ROS accumulation and inhibiting apoptotic cell death. Importantly, post-treatment with ALA consistently showed an almost equal restorative effect to pretreatment, in vitro and in vivo, supporting the possible use of ALA as a therapeutic agent for cisplatin-induced ototoxicity. This study is the first report on a strong therapeutic potential of ALA to rescue ototoxic hearing loss caused by cisplatin, and our data provide key evidence that ALA may act as a reducing agent for glutathione disulfide to increase glutathione levels on behalf of glutathione reductase. This result was consistent in both cultured cells and the mouse model, which improves the clinical value of ALA for therapy of cisplatin-induced ototoxicity.
Project description:Cisplatin-induced early-onset ototoxicity is linked to hearing loss. The mechanism by which cisplatin causes ototoxicity remains unclear. The purpose of this study was to identify the involvement of receptor-interacting protein kinase (RIP)3-dependent necroptosis in cisplatin-induced ototoxicity in vitro and in vivo. Sprague-Dawley rats (SD, 8 week) were treated via intraperitoneal (i.p.) injection with cisplatin (16 mg/kg for 1 day), and their hearing thresholds were measured by the auditory brainstem response (ABR) method. Hematoxylin and eosin (H & E) staining, immunohistochemistry, and western blots were performed to determine the effect of cisplatin-induced ototoxicity on cochlear morphology. Inhibitor experiments with necrostatin 1 (Nec-1) and Z-VAD were also performed in HEI-OC1 cell line. H&E stains revealed that the necroptotic changes were increased in the organ of Corti (OC) and spiral ganglion neurons (SGNs). Moreover, immunohistochemistry and western blot analysis showed that cisplatin treatment increased the protein levels of RIP3 in both OCs and SGNs. The treatment of Nec-1, a selective RIP1 inhibitor, resulted in markedly suppression of cisplatin-induced cell death in HEI-OC1 cells, whereas Z-VAD treatment did not change the cisplatin-induced cell death. Our results suggest that RIP3-dependent necroptosis was substantial in cisplatin-induced ototoxicity; inner cochlear regions, the OCs, and SGNs were especially sensitive to necroptosis.
Project description:In our previous study, we clearly demonstrated the roles of pro-inflammatory cytokines, including tumor necrosis factor-alpha, interleukin-1beta (IL-1beta), and IL-6, and subsequent reactive oxygen species (ROS) generation on the pathogenesis of cisplatin ototoxicity in vitro and in vivo. ROS generation in cisplatin-treated HEI-OC1 auditory cells was also correlated with changing mitochondrial membrane potential. However, the roles of NADPH oxidase in cisplatin-induced ROS generation and ototoxicity have not been fully elucidated. Herein, immunohistochemical studies demonstrated that treatment of cisplatin induced the expression of NADPH oxidase isoforms NOX-1 and NOX-4 in HEI-OC1 auditory cells. Expression of mRNA for NOX-1, NOX-4, NOXO1, NOXA1, p47(phox), and p67(phox) was also increased. Inhibition of NADPH oxidase with diphenyleniodonium chloride or apocynin abolished ROS production and the subsequent apoptotic cell death in cisplatin-treated cells. Furthermore, suppression of NOX1 and NOX4 expression by small interfering RNA transfection markedly abolished the cytotoxicity and ROS generation by cisplatin. Together, our data suggest that ROS generated, in part, through the activation of NADPH oxidase plays an essential role in cisplatin ototoxicity.
Project description:Recently, we demonstrated that pro-inflammatory cytokines such as TNF-alpha, IL-1beta, and IL-6 played a critical role in cisplatin-induced cochlear injury and that flunarizine, known as a T-type Ca(2+) channel antagonist, induced a cytoprotective effect against cisplatin cytotoxicity in HEI-OC1 cells by the activation of NF-E2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) cascade through PI3K-Akt signaling but calcium-independent pathway. We report here that flunarizine markedly attenuates cisplatin-induced pro-inflammatory cytokine secretion and their messenger RNA transcription as well as cisplatin cytotoxicity through the activation of Nrf2/HO-1 and downregulation of NF-kappaB. In HEI-OC1 cells, overexpression of Nrf2/HO-1 by gene transfer or pharmacological approaches attenuated cisplatin-induced cytotoxicity and pro-inflammatory cytokine production. On the contrary, inhibition of Nrf2/HO-1 signaling by pharmacological inhibitors or specific small interfering RNAs significantly abolished the beneficial effects of flunarizine. Flunarizine also attenuated cisplatin-mediated MAPK activation and pharmacological inhibition of MAPKs, especially MEK1/ERK, blocked cisplatin-induced NF-kappaB activation in HEI-OC1 cells. Furthermore, WT-Nrf2 overexpression effectively blocked MAPK activation after cisplatin exposure. Finally, orally administrated Sibelium, the trade name of flunarizine, suppressed the increase of pro-inflammatory cytokines by cisplatin in both serum and cochleas of mice, whereas it increased HO-1 expression in cochleas. These results indicate that flunarizine induces a protective effect against cisplatin ototoxicity through the downregulation of NF-kappaB by Nrf2/HO-1 activation and the resulting inhibition of pro-inflammatory cytokine production in vitro and in vivo.
Project description:Cisplatin is a highly-effective and widely-used chemotherapeutic agent that causes ototoxicity in many patients. Pharmacogenomic studies of key genes controlling drug biotransformation identified variants in thiopurine methyltransferase (TPMT) as predictors of cisplatin-induced ototoxicity, although the mechanistic basis of this interaction has not been reported. Expression constructs of TPMT*3A, *3B and *3C variants were generated and monitored in cultured cells. Cellular TPMT*3A levels were detected at >20-fold lower amounts than the wild type confirming the unstable nature of this variant. The expression of wild type TPMT (TPMT*1) in two murine ear cell lines, HEI-OC1 and UB/OC-1, significantly mitigated their susceptibility to cisplatin toxicity. Cisplatin treatment induced Tlr4 gene expression in HEI-OC1 cells and this response was blunted by the expression of wild type TPMT but not TPMT*3A. In line with the significant mitigation of TPMT*1-expressing cells to cisplatin cytotoxicity, these findings demonstrate a drug-gene interaction between increased TPMT activity and decreased susceptibility to cisplatin-induced toxicity of inner ear cells.
Project description:Eupatilin (5,7-dihydroxy-3',4',6-trimethoxyflavone) is a pharmacologically active flavone that has been isolated from a variety of medicinal plants and possesses a number of pharmacological properties. This study evaluates the antioxidant and antiapoptotic effects of eupatilin on cisplatin-induced ototoxicity using <i>in vitro</i> and <i>in vivo</i> models including HEI-OC1 cells, cochlear hair cells, and zebrafish. Employing a CCK8 assay and Annexin V-FITC/PI double staining, we found that eupatilin significantly alleviated cisplatin-induced apoptosis and increased hair cell viability. The level of reactive oxygen species (ROS) was evaluated by CellROX green and MitoSOX Red staining. The results showed that eupatilin possesses antioxidant activity. MitoTracker Red staining indicated that eupatilin remarkably decreased mitochondrial damage. Furthermore, we demonstrated that eupatilin protects hair cells from cisplatin-induced damage. Mechanistic studies in cisplatin-induced HEI-OC1 cells revealed that eupatilin promoted Bcl-2 expression, downregulated Bax expression, reversed the increase in caspase-3 and PARP activity, and reduced the expression of phosphorylated p38 and JNK. Our data suggest a novel role for eupatilin as a protective agent against ototoxic drug-induced hair cell apoptosis by inhibiting ROS generation and modulating mitochondrial-related apoptosis.
Project description:Cisplatin is a very effective chemotherapeutic, but severe and permanent hearing loss remains a prevalent side effect. The processes underpinning cisplatin-induced ototoxicity are not well understood. Gap junction channels composed of connexin (Cx) subunits allow for the passage of small molecules and ions between contacting neighboring cells. These specialized channels have been postulated to enhance cisplatin-induced cell death by spreading "death signals" throughout the supporting cells of the organ of Corti. This study sought to investigate the role of Cx43 in cisplatin-induced ototoxicity using organotypic cochlear cultures from control and two Cx43-mutant mouse strains harboring either a moderate (Cx43<sup>I130T/+</sup>) or severe (Cx43<sup>G60S/+</sup>) reduction of Cx43 function. Cochlear cultures from Cx43-mutant mice with a severe reduction in Cx43-based gap junctional intercellular communication (GJIC) had an enhanced number of hair cells that were positive for cleaved caspase 3, a marker of active apoptosis, after cisplatin treatment. In cisplatin-treated organotypic cochlear cultures, there was a decrease in the co-localization of Cx26 and Cx30 compared with untreated cultures, suggesting that cisplatin causes reorganization of connexin composition in supporting cells. Both Cx26 and Cx30 protein expression as well as GJIC were decreased in organotypic cochlear cultures treated with the gap-junction blocker carbenoxolone. When cisplatin and carbenoxolone were co-administered, there were no differences in hair cell loss compared with cisplatin treatment alone. Using cisplatin-treated control and Cx43-ablated organ of Corti derived HEI-OC1 mouse cells, we found that greatly reducing GJIC led to preferential induction of an ER stress pathway. Taken together, this study strongly suggests that inhibition of GJIC in organ of Corti cells does not lead to differential susceptibility to cisplatin-induced ototoxicity. Although cisplatin causes the same degree of cell death in gap junction competent and incompetent cochlear cells, the engagement of the mitochondrial dysregulation and ER stress differs.