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 Corti's 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.

Potential anticancer properties of the water extract of Inonotus [corrected] 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.

Sasim attenuates LPS-induced TNF-alpha production through the induction of HO-1 in THP-1 differentiated macrophage-like cells.

AIM OF THE STUDY: Sasim, a traditional prescription composed of seven herbal mixtures, has been widely used as an oriental medicine for the treatment of cerebral infarction in Korea. However, the regulatory mechanisms by which the formula affects immune processing in cerebral infarction patients remain unknown. MATERIALS AND METHODS: The levels of secretory protein of tumor necrosis factor (TNF)-alpha were determined in both THP-1 differentiated macrophage-like (THP-1/M) cells and Peripheral blood mononuclear cells (PBMCs) from cerebral infarction patients. Also, the levels of protein and mRNA of TNF-alpha and heme oxygenase-1 (HO-1) were detected in THP-1/M cells under our experimental condition. RESULTS: Sasim markedly suppressed lipopolysaccharide (LPS)-induced TNF-alpha at the levels of secretory protein and mRNA in both PBMCs from cerebral infarction patients and THP-1/M cells. Interestingly, Sasim strongly induced HO-1, the rate-limiting enzyme of heme catabolism, at both the protein and mRNA levels in THP-1/M cells. Treatment with tin protoporphyrin IX (SnPP), an inhibitor of the catalytic activity of HO, significantly abolished the suppressive effect of Sasim on LPS-induced TNF-a production in THP-1/M cells. CONCLUSIONS: These data indicate that Sasim may be beneficial in the cessation of inflammatory processes associated with cerebral infarction through the induction of HO-1 expression.

Role of proinflammatory cytokines in cisplatin-induced vestibular hair cell damage.

BACKGROUND: Cisplatin causes the impairment of inner ear functions, including hearing and balance, through the involvement of a number of mechanisms. However, no laboratory studies have been performed on involvement of inflammation-related events in cisplatin-mediated vestibular dysfunction. METHODS: We evaluated the secretion of proinflammatory cytokines and nuclear factor-kappaB (NF-kappaB) activation in cisplatin-treated UB/UE-1 utricular epithelial cells. We also employed immunohistochemistry to detect proinflammatory cytokines and NF-kappaB expression in cisplatin-injected mice. RESULTS: Productions of proinflammatory cytokines significantly caused the death of UB/UE1 cells by cisplatin. Pharmacologic inhibition of mitogen-activated protein (MAP) kinase/ERK kinase-1 (MEK1) or extracellular signal-regulated kinase (ERK) significantly attenuated the death of UB/UE1 cells caused by cisplatin and proinflammatory cytokines. Immunohistochemical studies revealed an increase in the expression of proinflammatory cytokines and NF-kappaB in both the cristae ampullae and utricle of cisplatin-injected mice. CONCLUSIONS: These results suggest that proinflammatory cytokines may play an important role in the pathogenesis of cisplatin-mediated vestibulo-toxicity.

Berberine, a natural product, combined with cisplatin enhanced apoptosis through a mitochondria/caspase-mediated pathway in HeLa cells.

Berberine, a main component of Coptidis Rhizoma, has been extensively studied and is known to exhibit multiple pharmacologic activities. In this study, we investigated whether the combination of berberine and cisplatin exhibited significant cytotoxicity in HeLa cells. Apoptosis was evaluated based on DNA fragmentation and cytofluorometrically with the annexin-V/propidium iodide labeling method. Combined treatment with berberine and cisplatin acted in concert to induce loss of mitochondrial membrane potential (Delta Psi m), release of cytochrome-c from mitochondria, and decreased expression of antiapoptotic Bcl-2, Bcl-x/L, resulting in activation of caspases and apoptosis. Further study showed that cell death induced by the combined treatment was associated with increased reactive oxygen species generation and lipid peroxidation. Moreover, we discovered that the combined treatment-induced apoptosis was mediated by the activation of the caspase cascade. These results indicated that the potential of cytotoxicity mediated through the mitochondria-caspase pathway is primarily involved in the combined treatment-induced apoptosis.

Antiinflammatory effect of Daesiho, a Korean traditional prescription for cerebral infarct patients.

Daesiho, a prescription composed of eight herbal mixtures, has been widely used in the treatment of cerebral infarct in Oriental medicine. However, the mechanisms by which the formula affects the production of pro-inflammatory cytokines in cerebral infarct patients remains unknown. The levels of secretory protein pro-inflammatory cytokines, including tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta and IL-6, were significantly increased in both lipopolysaccharide (LPS) and phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMCs) from cerebral infarct patients and LPS-stimulated THP-1 differentiated macrophage-like cells (THP-1/M). However, pretreatment with Daesiho significantly inhibited the secretion of pro-inflammatory cytokines, including TNF-alpha, IL-1beta, and IL-6, in stimulated PBMCs and THP-1/M cells. In addition, Daesiho significantly suppressed mRNA expression of pro-inflammatory cytokines. Therefore, these data indicate that Daesiho may be beneficial in the cessation of inflammatory processes of cerebral infarction through suppression of the production of pro-inflammatory cytokines via inhibition of mRNA expression.

Berberine induces G1 arrest and apoptosis in human glioblastoma T98G cells through mitochondrial/caspases pathway.

Berberine is an isoquinoline plant alkaloid with a long history of being used for the treatment of many diseases in Chinese herbal medicine. Berberine has a wide range of biochemical and pharmacological effects, including antitumor activities, but its mechanism of action is not clearly understood. In this study, we investigated that the relationship between the antiproliferative activities of berberine and the apoptotic pathway associated with its molecular mechanism of action in human glioblastoma T98G cells. Berberine treatment of T98G cell lines inhibited cell proliferation and induced cell death in a dose (50-200 microg/ml) dependent manner with an IC50 value of 134 microg/ml, which was associated with an increase in G1 arrest. Western blot analysis showed that the berberine-induced G1 arrest was mediated through the increased expression of P27 and the decreased expression of cyclin-dependent kinase (CDK) 2, CDK4, cyclin D, and cyclin E proteins. Berberine treatment also markedly enhanced apoptosis in T98G cells through the induction of a higher ratio of the Bax/Bcl-2 proteins, the disruption of mitochondrial membrane potential, and the activation of procaspase-9, caspase-9, caspase-3, and poly(ADP-ribose) polymerase (PARP). Berberine can inhibit T98G cell proliferation by inducing G1 arrest and apoptosis. These results demonstrate that the berberine-induced apoptosis of T98G cells is primarily mediated through the mitochondrial/caspases-dependent pathway.

Chaga mushroom (Inonotus obliquus) induces G0/G1 arrest and apoptosis in human hepatoma HepG2 cells.

AIM: To investigate the anti-proliferative and apoptotic effects of Chaga mushroom (Inonotus obliquus) water extract on human hepatoma cell lines, HepG2 and Hep3B cells. METHODS: The cytotoxicity of Chaga extract was screened by 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay. Morphological observation, flow cytometry analysis, Western blot were employed to elucidate the cytotoxic mechanism of Chaga extract. RESULTS: HepG2 cells were more sensitive to Chaga extract than Hep3B cells, as demonstrated by markedly reduced cell viability. Chaga extract inhibited the cell growth in a dose-dependent manner, which was accompanied with G0/G1-phase arrest and apoptotic cell death. In addition, G0/G1 arrest in the cell cycle was closely associated with down-regulation of p53, pRb, p27, cyclins D1, D2, E, cyclin-dependent kinase (Cdk) 2, Cdk4, and Cdk6 expression. CONCLUSION: Chaga mushroom may provide a new therapeutic option, as a potential anticancer agent, in the treatment of hepatoma.

Suppression of Nrf2-driven heme oxygenase-1 enhances the chemosensitivity of lung cancer A549 cells toward cisplatin.

Heme oxygenase-1 (HO-1) is highly expressed in various tumor tissues and plays an important role in tumor cell growth through anti-oxidative and anti-apoptotic effects. Herein, we demonstrate that A549 cells express high levels of HO-1, Nrf2, and NF-kappaB compared to other lung cancer cell lines, including H23, H157, and H460. Ectopic expression of HO-1 small interfering RNA (siRNA) increased both apoptosis and degradation of procaspase-3. Transfection studies with siRNA specific for Nrf2 and NF-kappaB revealed that HO-1 expression in A549 cells is mediated by transcriptional activation of Nrf2, but not NF-kappaB. A549 cells are less susceptible to cisplatin cytotoxicity than other lung cancer cell lines, concomitant with increases in HO-1 expression and MAPK phosphorylation in a time-dependent fashion. Furthermore, inhibition of HO-1 by siRNA and a specific HO-1 inhibitor ZnPP augments cisplatin cytotoxicity toward A549 cells. Pharmacologic suppression of HO-1 activity resulted in a marked increase in the ROS generation in cisplatin-treated cells. In addition, pharmacologic inhibitors of MAPK suppressed the induction of HO-1 and Nrf2 expression by cisplatin. These findings suggest that HO-1 may modulate the chemosensitivity of lung cancer A549 cells to cisplatin through the MAPK-Nrf2 pathway.

Anti-inflammatory effect of So-Pung-Tang, a Korean traditional prescription for cerebral infarction patients.

So-Pung-Tang (Sopung), a prescription composed of 14 herbal mixtures, has been widely used in the treatment of cerebral infarction in Oriental Medicine. However, the mechanisms by which the formula affects on the production of pro-inflammatory cytokines in cerebral infarction patients remain unknown yet. The levels of secretory protein of pro-inflammatory cytokines, including tumor necrosis factor (TNF)-alpha, interlukin (IL)-1beta, and IL-6, were significantly increased in both THP-1 differentiated macrophage-like cells (THP-1/M) and peripheral blood mononuclear cells (PBMCs) from cerebral infarction patients after stimulation. However, pretreatment with Sopung markedly inhibited the secretion of TNF-alpha and IL-6, but not IL-1beta, in lipopolysaccharide (LPS)-stimulated THP-1/M cells and PBMCs treated with LPS and phytohemagglutinin (PHA). Furthermore, Sopung significantly inhibited LPS-induced phosphorylation of extracellular signal-regulated kinase (ERK1/2) and c-jun N-terminal kinase (JNK), but not p38 in THP-1/M cells. These data indicate that Sopung may be beneficial in the cessation of inflammatory processes of cerebral infarction through suppression of ERK1/2 and JNK activation.

Hibiscus sabdariffa L. water extract inhibits the adipocyte differentiation through the PI3-K and MAPK pathway.

Hibiscus sabdariffa L., a tropical beverage material and medical herb, is used commonly as in folk medicines against hypertension, pyrexia, inflammation, liver disorders, and obesity. This report was designed to investigate the inhibitory mechanisms of hibiscus extract on adipocyte differentiation in 3T3-L1 preadipocytes. The possible inhibitory pathways that regulate the adipocyte differentiation contain the adipogenic transcription factors, C/EBPalpha and PPARgamma, PI3-kinase, and MAPK pathway. In this study, we examined whether hibiscus extract affected the adipogenesis via these three pathways. To differentiate preadipocyte in adipocyte, confluent 3T3-L1 preadipocytes were treated with the hormone mixture including isobutylmethylxanthine, dexamethasone, and insulin (MDI). Hibiscus extract inhibited significantly the lipid droplet accumulation by MDI in a dose-dependent manner and attenuated dramatically the protein and mRNA expressions of adipogenic transcriptional factors, C/EBPalpha and PPARgamma, during adipogenesis. The increase of phosphorylation and expression of PI3-K/Akt during adipocytic differentiation was markedly inhibited by treatment with hibiscus extract or PI3-K inhibitors. Furthermore, the phosphorylation and expression of MEK-1/ERK known to regulate the early phase of adipogenesis were clearly decreased with the addition of hibiscus extract. Taken together, this report suggests that hibiscus extract inhibits the adipocyte differentiation through the modulation of PI3-K/Akt and ERK pathway that play pivotal roles during adipogenesis.

Anti-inflammatory effect of Sasim extracts in PHA-stimulated THP-1 and peripheral blood mononuclear cells from cerebral infarction patients.

Sasim, a prescription composed of seven herbal mixtures, has been widely used for the treatment of cerebral infarction as an oriental medicine in Korea. However, the mechanisms by which the formula affects on the production of pro-inflammatory cytokines in cerebral infarct patients remain unknown yet. The levels of secretory protein and mRNA of pro-inflammatory cytokines, including tumor necrosis factor (TNF)-alpha, interlukin (IL)-1beta, and IL-6, were significantly increased in both THP-1 differentiated macrophage-like cells (T/M) and peripheral blood mononuclear cells (PBMCs) from cerebral infarct patients at 24h after stimulation with phytohemagglutinin (PHA) (p<0.05). However, pretreatment of Sasim strongly suppressed the secretion of pro-inflammatory cytokines in PHA-stimulated T/M cells and PBMCs. Moreover, Sasim significantly suppressed the transcriptional levels of pro-inflammatory cytokines in PHA-stimulated THP-1/M cells. These data indicate that Sasim may be beneficial in the cessation of inflammatory processes of cerebral infarction through suppression on the production of pro-inflammatory cytokines via inhibition of mRNA expression.

Samul extract protects against the H2O2-induced apoptosis of H9c2 cardiomyoblasts via activation of extracellular regulated kinases (Erk) 1/2.

Samul extract, containing Radix Rehmanniae, Radix Angelicae Gigantis, Radix Paeoniae, and Rhizoma Cnidii, has been traditionally used for treatment of ischemic heart and brain damages in Oriental medicine. However, little is known about the mechanism by which Samul rescues cells from cytotoxic damage. This study was designed to investigate the protective mechanisms of Samul on H(2)O(2)-induced death of H9c2 cells. Treatment with H(2)O(2) markedly decreased the viability of H9c2 cells in a dose- and time-dependent manner, which was significantly prevented by pre-treatment with Samul. The nature of death of H9c2 cells by H(2)O(2) was demonstrated by apoptotic features, including ladder-pattern fragmentation of genomic DNA and chromatin condensation, which were markedly abolished by pretreatment of Samul in H(2)O(2)-treated cells. We further demonstrated that MEK inhibitor, PD98059, dose-dependently attenuated the protective effects of Samul against H(2)O(2), whereas inhibitors of Jnk and p38 did not. Consistently, Samul induced the early phosphorylation of Erk, p44, in H(2)O(2)-treated cells. In addition, treatment with Samul also resulted in an increase of expression of anti-apotogenic Bcl2 protein, which was decreased by H(2)O(2). However, it inhibited the expression of apotogenic Bax protein in H(2)O(2)-treated cells. Taken together, these results suggest that the protective effects of Samul against oxidative damage may be achieved via activation of MAP kinase, Erk as well as Bcl2 family proteins.

Combination treatment with arsenic trioxide and sulindac augments their apoptotic potential in lung cancer cells through activation of caspase cascade and mitochondrial dysfunction.

Non-steroidal anti-inflammatory drugs (NSAIDs) are known to enhance the responsiveness of tumor cells toward chemotherapeutic drugs and radiation. However, the precise mechanism of synergistic enhancement in tumoricidal activity is not clearly known. Herein, we demonstrate that the combination treatment of arsenic trioxide (As2O3) and sulindac resulted in a synergistic augmentation of cytotoxicity toward NCI-H157 lung cancer cells, which was revealed as apoptosis accompanied by chromatin fragmentation and an increase in sub-G0/G1 fraction. In addition, combination treatment with As2O3 and sulindac increased the catalytic activity of caspase-3, -8, and -9 along with induction of Fas/FasL expression and cytosolic release of cytochrome c. Pharmacologic scavenging study of reactive oxygen species (ROS) revealed that synergistic augmentation of cytotoxicity was achieved by generation of ROS, which might modulate the expression of Bcl-2 family proteins, the activity of caspase-3, and mitochondrial membrane potential transition.

Heme oxygenase-1 attenuates the cisplatin-induced apoptosis of auditory cells via down-regulation of reactive oxygen species generation.

Heme oxygenase-1 (HO-1), the rate-limiting enzyme of heme catabolism, is known to modulate various cellular functions, including cytokine production, cell proliferation, and apoptosis, in stress-related conditions. However, the role of HO-1 in the auditory system remains elusive. Herein, we demonstrate that pharmacologic induction of HO-1 along with catalytic activation significantly suppressed apoptosis of HEI-OC1 cells induced by cisplatin. Studies of ectopic expression of pcDNA3-HO-1 and siRNA of HO-1 further revealed the protective role of HO-1 against cisplatin in HEI-OC1 cells. Among the catabolic metabolites of HO-1, both carbon monoxide (CO) and bilirubin were directly involved in the protective role of HO-1 against cisplatin through inhibition of reactive oxygen species generation. Furthermore, pharmacological induction of HO-1 completely prevented the destruction of outer hair cell arrays by cisplatin through a CO-dependent mechanism in organotrophic culture of the rat primary organ of Corti explants. These results suggest that HO-1 may serve as a safeguard of auditory sensory hair cells against a variety of challenges of oxidative stress, including noise trauma, presbycusis, and ototoxic drugs, respectively.

Trichostatin A induces apoptosis in lung cancer cells via simultaneous activation of the death receptor-mediated and mitochondrial pathway?

Trichostatin A (TSA), originally developed as an antifungal agent, is one of potent histone deacetylase (HDAC) inhibitors, which are known to cause growth arrest and apoptosis induction of transformed cells, including urinary bladder, breast, prostate, ovary, and colon cancers. However, the effect of HDAC inhibitors on human non-small cell lung cancer cells is not clearly known yet. Herein, we demonstrated that treatment of TSA resulted in a significant decrease of the viability of H157 cells in a dose-dependent manner, which was revealed as apoptosis accompanying with nuclear fragmentation and an increase in sub-G0/G1 fraction. In addition, it induced the expression of Fas/FasL, which further triggered the activation of caspase-8. Catalytic activation of caspase-9 and decreased expression of anti-apoptotic Bcl-2 and Bcl-XL proteins were observed in TSA-treated cells. Catalytic activation of caspase-3 by TSA was further confirmed by cleavage of pro-caspase-3 and intracellular substrates, including poly (ADP-ribose) polymerase (PARP) and inhibitor of caspase-activated deoxyribonuclease (ICAD). In addition, a characteristic phenomenon of mitochondrial dysfunction, including mitochondrial membrane potential transition and release of mitochondrial cytochrome c into the cytosol was apparent in TSA-treated cells. Taken together, our data indicate that inhibition of HDAC by TSA induces the apoptosis of H157 cells through signaling cascade of Fas/FasL-mediated extrinsic and mitochondria-mediated intrinsic caspases pathway.