Yunha Kim

Cisplatin Cytotoxicity of Auditory Cells Requires Secretions of Proinflammatory Cytokines via Activation of ERK and NF-κB

The ototoxicity of cisplatin, a widely used chemotherapeutic agent, involves a number of mechanisms, including perturbation of redox status, increase in lipid peroxidation, and formation of DNA adducts. In this study, we demonstrate that cisplatin increased the early immediate release and de novo synthesis of proinflammatory cytokines, including TNF-α, IL-1β, and IL-6, through the activation of ERK and NF-κB in HEI-OC1 cells, which are conditionally immortalized cochlear cells that express hair cell markers. Both neutralization of proinflammatory cytokines and pharmacologic inhibition of ERK significantly attenuated the death of HEI-OC1 auditory cells caused by cisplatin and proinflammatory cytokines. We also observed a significant increase in the protein and mRNA levels of proinflammatory cytokines in both serum and cochleae of cisplatin-injected rats, which was suppressed by intraperitoneal injection of etanercept, an inhibitor of TNF-α. Immunohistochemical studies revealed that TNF-α expression was mainly located in the spiral ligament, spiral limbus, and the organ of Corti in the cochleae of cisplatin-injected rats. NF-κB protein expression, which overlapped with terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling-positive signal, was very strong in specific regions of the cochleae, including the organ of Corti, spiral ligament, and stria vascularis. These results indicate that proinflammatory cytokines, especially TNF-α, play a central role in the pathophysiology of sensory hair cell damage caused by cisplatin.

Evidence that Cisplatin-induced Auditory Damage is Attenuated by Downregulation of Pro-inflammatory Cytokines Via Nrf2/HO-1

Recently, we demonstrated that pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6 played a critical role in cisplatin-induced cochlear injury and that flunarizine, known as a T-type Ca2+ 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-κB. 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-κB 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-κB by Nrf2/HO-1 activation and the resulting inhibition of pro-inflammatory cytokine production in vitro and in vivo.

Ebselen attenuates cisplatin-induced ROS generation through Nrf2 activation in auditory cells.

Ebselen, an organoselenium compound that acts as a glutathione peroxidase mimetic, has been demonstrated to possess antioxidant and anti-inflammatory activities. However, the molecular mechanism underlying this effect is not fully understood in auditory cells. The purpose of the present study is to investigate the protective effect of ebselen against cisplatin-induced toxicity in HEI-OC1 auditory cells, organotypic cultures of cochlear explants from two-day postnatal rats (P(2)) and adult Balb/C mice. Pretreatment with ebselen ameliorated apoptotic death induced by cisplatin in HEI-OC1 cells and organotypic cultures of Cortis organ. Ebselen pretreatment also significantly suppressed cisplatin-induced increases in intracellular reactive oxygen species (ROS), intracellular reactive nitrogen species (RNS) and lipid peroxidation levels. Ebselen dose-dependently increased the expression level of an antioxidant response element (ARE)-luciferase reporter in HEI-OC1 cells through the translocation of Nrf2 into the nucleus. Furthermore, we found that pretreatment with ebselen significantly restored Nrf2 function, whereas it ameliorated the cytotoxicity of cisplatin in cells transfectants with either a pcDNA3.1 (control) or a DN-Nrf2 (dominant-negative) plasmid. We also observed that Nrf2 activation by ebselen increased the expression of phase II antioxidant genes, including heme oxygenase (HO-1), NAD(P)H:quinine oxidoreductase, and gamma-glutamylcysteine synthetase (gamma-GCS). Treatment with ebselen resulted in an increased expression of HO-1 and intranuclear Nrf2 in hair cells of organotypic cultured cochlea. After intraperitoneal injection with cisplatin, auditory brainstem responses (ABRs) threshold was measured on 8th day in Balb/C mice. ABR threshold shift was marked occurred in mice injected with cisplatin (16 mg/kg, n=5; Click and 8-kHz stimuli, p<0.05; 4, 16 and 32 kHz, p<0.01), whereas that of animal group which was treated with cisplatin and ebselen was not significantly changed. These results suggest that ebselen activates the Nrf2-ARE signaling pathway, which ultimately prevents free radical stresses from cisplatin and further contributes to protect auditory sensory hair cells from free radicals produced by cisplatin.

Sedative activity of two flavonol glycosides isolated from the flowers of Albizzia julibrissin durazz

The flowers of Albizzia julibrissin are used as a sedative in oriental traditional medicine. The phytochemical study of this plant allowed the isolation of two flavonol glycosides, quercitrin (1) and isoquercitrin (2). The sedative activity of these compounds was evaluated, and both compounds 1 and 2 increased pentobarbital-induced sleeping time in dose-dependent manner in mice. These results support the use of the flowers of this plant as a sedative agent.

Isoliquiritigenin, from Dalbergia odorifera, up-regulates anti-inflammatory heme oxygenase-1 expression in RAW264.7 macrophages.

Abstract.Objectives:Isoliquiritigenin (ISL), one of the major constituents of Dalbergia odorifera T. Chen (Leguminosae), is reported to exert anti-inflammatory effects, but the relevant anti-inflammatory mechanisms are not completely understood. Heme oxygenase-1 (HO-1) has been proven to be involved in the resolution of inflammatory responses. In this study, we investigated whether ISL could induce HO-1 expression in RAW264.7 macrophages, and if so, whether HO-1 could mediate the anti-inflammatory effects of ISL.Methods:The protein expression of inducible nitric oxide synthase and HO-1 was analyzed by western blot analysis. The production of nitric oxide (NO) and interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) was assayed by Griess and ELISA, respectively. The TNF-α and HO-1 mRNA expression was analyzed by northern blot analysis.Results:ISL markedly suppressed LPS-induced NO, IL-1β, and TNF-α production. ISL induced HO-1 expression through the extracellular signal-regulated kinase1/2 pathway in RAW264.7 macrophages. The effects of ISL on LPS-induced NO and TNF-α production were reversed by the HO-1 inhibitor, tin protoporphyrin.Conclusions:ISL is an effective HO-1 inducer capable of inhibiting macrophage-derived inflammation.

Effect of the aqueous extract of Aquilaria agallocha stems on the immediate hypersensitivity reactions

We investigated the effects of the aqueous extract of Aquilaria agallocha Roxb. (Thymelaeaceae) on the immediate hypersensitivity reactions. The aqueous extract of Aquilaria agallocha stems showed inhibitory effects on passive cutaneous anaphylaxis, anaphylaxis induced by compound 48/80, and histamine release from rat peritoneal mast cells (RPMC). The morphological examination also clearly showed that the extract prevented the degranulation of RPMC in rats. The level of compound 48/80-induced intracellular cAMP in RPMC, when the extract was added, significantly increased about 8-fold at 10 s compared with that of basal cells. These results suggest that the aqueous extract of Aquilaria agallocha stems inhibits the immediate hypersensitivity reaction by inhibition of histamine release from mast cells.

Potential anticancer properties of the water extract of Inontus obliquus by induction of apoptosis in melanoma B16-F10 cells

ETHNOPHARMACOLOGICAL RELEVANCE Inonotus obliquus (Chaga mushroom), one of the widely known medicinal mushrooms, has been used to treat various cancers in Russia and most of Baltic countries for many centuries. AIM OF THE STUDY To examine the anti-proliferative effects of Inonotus obliquus extract on melanoma B16-F10 cells. Furthermore, to assess the anti-tumor effect of Inonotus obliquus extract in vivo in Balb/c mice. MATERIALS AND METHODS The water extract of Inonotus obliquus was studied for anti-proliferative effects on the growth and morphology of B16-F10 melanoma cells and for anti-tumor effect using in vivo in Balb/c mice. RESULTS Inonotus obliquus extract not only inhibited the growth of B16-F10 cells by causing cell cycle arrest at G(0)/G(1) phase and apoptosis, but also induced cell differentiation. These effects were associated with the down-regulation of pRb, p53 and p27 expression levels, and further showed that Inonotus obliquus extract resulted in a G(0)/G(1) cell cycle arrest with reduction of cyclin E/D1 and Cdk 2/4 expression levels. Furthermore, the anti-tumor effect of Inonotus obliquus extract was assessed in vivo in Balb/c mice. Intraperitoneal administration of Inonotus obliquus extract significantly inhibited the growth of tumor mass in B16-F10 cells implanted mice, resulting in a 3-fold (relative to the positive control, (*)p<0.05) inhibit at dose of 20mg/kg/day for 10 days. CONCLUSION This study showed that the water extract of Inonotus obliquus mushroom exhibited a potential anticancer activity against B16-F10 melanoma cells in vitro and in vivo through the inhibition of proliferation and induction of differentiation and apoptosis of cancer cells.

Antiproliferative effects of alkaloids from Sedum sarmentosum on murine and human hepatoma cell lines.

The whole plant of Sedum sarmentosum (SS) has been traditionally used for the treatment of chronic viral hepatitis in China and South Korea. Certain hepatitis virus causes acute and chronic hepatitis and induces hepatocellular carcinoma (HC). In the present study, we examined whether the crude alkaloid fraction (CAF) of SS had any anticancer effects on hepatoma cell lines. Murine hepatoma (BNL CL. 2) and human hepatoma (HepG2) cell lines were cultured in the presence of CAF of SS at various doses (50-150 microg/ml) for 24 or 48 h. CAF caused a dose-dependent inhibition of cell proliferation without necrosis or apoptosis. Antiproliferative effects of CAF of SS were associated with an increase in the number of cells in the G1 phase of cell cycle. This study suggests that SS may improve survival of hepatoma patients via the inhibition of excessive growth of tumor cells.

Costunolide inhibits production of tumor necrosis factor-α and interleukin-6 by inducing heme oxygenase-1 in RAW264.7 macrophages

Abstract.Objectives:Heme oxygenase (HO)-1 expression via nuclear factor-erythroid 2-related factor 2 (Nrf2) activation has an ability to inhibit tumor necrosis factor (TNF)-α and interleukin (IL)-6 production. Costunolide has been reported to inhibit IL-1 production, but whether other cytokines could be inhibited remains to be confirmed. We investigated the effects of costunolide and its components (α-methylene-γ-butyrolactone; CH2-BL, α-methyl-γ-butyrolactone; CH3-BL, and γ-butyrolactone; BL) on HO-1 expression as well as TNF-α and IL-6 production in RAW264.7 macrophages.Methods:HO-1 expression and Nrf2 nuclear accumulation were analyzed by Western blot analysis. The production of TNF-α and IL-6 in RAW264.7 macrophages stimulated with lipopolysaccharide (LPS) was assayed by ELISA.Results:Costunolide and CH2-BL induced HO-1 expression and Nrf2 nuclear accumulation, whereas CH3-BL and BL did not. Pre-incubation with costunolide inhibited LPS-induced production of TNF-α and IL-6. The inhibitory effects of costunolide on TNF-α and IL-6 production were abrogated by tin protoporphyrin, an HO inhibitor.Conclusions:Costunolide is an effective HO-1 inducer capable of inhibiting macrophage-derived pro-inflammatory cytokines. CH2-BL moiety of costunolide is essential for Nrf2 activation leading to HO-1 expression.

Activation of Lipopolysaccharide–TLR4 Signaling Accelerates the Ototoxic Potential of Cisplatin in Mice

Dysfunction in immune surveillance during anticancer chemotherapy of patients often causes weakness of the host defense system and a subsequent increase in microbial infections. However, the deterioration of organ-specific function related to microbial challenges in cisplatin-treated patients has not yet been elucidated. In this study, we investigated cisplatin-induced TLR4 expression and its binding to LPS in mouse cochlear tissues and the effect of this interaction on hearing function. Cisplatin increased the transcriptional and translational expression of TLR4 in the cochlear tissues, organ of Corti explants, and HEI-OC1 cells. Furthermore, cisplatin increased the interaction between TLR4 and its microbial ligand LPS, thereby upregulating the production of proinflammatory cytokines, such as TNF-α, IL-1β, and IL-6, via NF-κB activation. In C57BL/6 mice, the combined injection of cisplatin and LPS caused severe hearing impairment compared with that in the control, cisplatin-alone, or LPS-alone groups, whereas this hearing dysfunction was completely suppressed in both TLR4 mutant and knockout mice. These results suggest that hearing function can be easily damaged by increased TLR expression and microbial infections due to the weakened host defense systems of cancer patients receiving therapy comprising three to six cycles of cisplatin alone or cisplatin combined with other chemotherapeutic agents. Moreover, such damage can occur even though patients may not experience ototoxic levels of cumulative cisplatin concentration.