Hyeonseok Ko

Hyperoside protects primary rat cortical neurons from neurotoxicity induced by amyloid β-protein via the PI3K/Akt/Bad/BclXL-regulated mitochondrial apoptotic pathway

Amyloid β-protein (Aβ), which is deposited in neurons as neurofibrillary tangles, is known to exert cytotoxic effects by inducing mitochondrial dysfunction. Additionally, the PI3K/Akt-mediated interaction between Bad and Bcl(XL) plays an important role in maintaining mitochondrial integrity. However, the application of therapeutic drugs, especially natural products in Alzheimers disease therapy via PI3K/Akt/Bad/Bcl(XL)-regulated mitochondrial apoptotic pathway has not aroused extensive attention. In the present study, we investigated the neuroprotective effects of hyperoside, a bioactive flavonoid compound from Hypericum perforatum, on Aβ(25-35)-induced primary cultured cortical neurons, and also examined the potential cellular signaling mechanism for Aβ detoxication. Our results showed that treatment with hyperoside significantly inhibited Aβ(25-35)-induced cytotoxicity and apoptosis by reversing Aβ-induced mitochondrial dysfunction, including mitochondrial membrane potential decrease, reactive oxygen species production, and mitochondrial release of cytochrome c. Further study indicated that hyperoside can activate the PI3K/Akt signaling pathway, resulting in inhibition of the interaction between Bad and Bcl(XL), without effects on the interaction between Bad and Bcl-2. Furthermore, hyperoside inhibited mitochondria-dependent downstream caspase-mediated apoptotic pathway, such as that involving caspase-9, caspase-3, and poly ADP-ribose polymerase (PARP). These results demonstrate that hyperoside can protect Aβ-induced primary cultured cortical neurons via PI3K/Akt/Bad/Bcl(XL)-regulated mitochondrial apoptotic pathway, and they raise the possibility that hyperoside could be developed into a clinically valuable treatment for Alzheimers disease and other neuronal degenerative diseases associated with mitochondrial dysfunction.

Icariin attenuates β-amyloid-induced neurotoxicity by inhibition of tau protein hyperphosphorylation in PC12 cells.

Alzheimers disease (AD) is a neurodegenerative disease characterized by the progressive loss of neurons and production of β-amyloid proteins (Aβ). Hyperphosphorylation of tau protein is proposed to be an early event for the evolution of AD, and may play an important role in Aβ-induced neurodegeneration. Icariin, a flavonoid compound from the herb Epimedium brevicornum Maxim, exerts a protective effect on learning and memory abilities in Aβ(25-35)-induced AD rats. However, the molecular mechanism of icariin-induced neuroprotective effect against tau protein hyperphosphorylation, which is one of the most representative hallmarks in AD, is still unknown. In the present study, we investigated the inhibitory effect of icariin on Aβ(25-35)-induced tau protein hyperphosphorylation on PC12 cells. The results showed that treatment with icariin significantly decreased Aβ(25-35)-induced cytotoxicity and apoptosis rate through inhibiting tau protein hyperphosphorylation at Ser396, Ser404 and Thr205 sites, respectively. Mechanism study showed that icariin could activate PI3K/Akt signaling pathway, resulting in an inhibitory effect on glycogen synthase kinase (GSK)-3β, which is an important kinase response for tau protein hyperphosphorylation in the development of AD. These observations indicate that icariin is capable of attenuating Aβ(25-35)-induced tau protein hyperphosphorylation and promoting survival of neuronal cells, meanwhile also provide some insights into the potential signaling pathway that is involved. Thus, this study promises a great potential agent for Alzheimers disease and other tau pathology-related neuronal degenerative diseases.

The neuroprotective effect of modified Yeoldahanso-tang via autophagy enhancement in models of Parkinson's disease

AIM OF STUDY Modified Yeoldahanso-tang (MYH) is a Korean herbal formula, containing 10 herbs: Pueraria lobata (Willd.) Ohwi, Angelica tenuissima Nakai, Scutellaria baicalensis Georgi, Platycodon grandiflorum (Jacq), Angelicae Dahurica, Cimicifuga heracleifolia Kom, Raphanus sativa L., Polygala tenuifolia (Willd.), Acorus gramineus Soland. and Dimocarpus longan Lour. The constitutive ratio of the ten herbs is at 6:4:2:1:2:2:2:4:6:6 in dry weight. MYH has been used to treat amnesia, hypochondria and dementia in Korea. In this study, we explored the possibility of using MYH in the prevention and treatment of Parkinsons disease (PD). Specifically, we made an effort to demonstrate the neuroprotective effects of MYH using experimental methods similar to those used in a recent study of PD. MATERIALS AND METHODS 1-Methyl-4-phenylpyridinium (MPP+) (400μM) was used to induce cytotoxicity in NGF (nerve growth factor)-differentiated PC12 cells. Cell viability was measured using a MTT assay. Induction of autophagy by MYH in NGF-differentiated PC12 cells was measured using an immunoblotting assay with LC3 and beclin 1 antibodies. The proteasomal inhibitor lactacystin (10μM) was used to cause UPS dysfunction in NGF-differentiated PC12 cells. Clearance of aggregated proteins by MYH was measured using an immunoblotting assay with an ubiquitin antibody. 1-Methyl-4-phenyl-1,2,3,6-tetrahydrophenylpyridine (MPTP) (20mg/kg, 4 times i.p.) caused substantia nigra injuries in C57BL/6 mice. Dopamine (DA) neurons were identified using a tyrosine hydroxylase-immunohistochemistry (TH-IHC) assay with a rabbit anti-TH antibody. RESULTS Our findings indicate that MYH provides protection against MPP+-induced injury in NGF-differentiated PC12 cell. And MYH provides neuroprotection against lactacystin-induced NGF-differentiated PC12 cell death, which effect is partially mediated by autophagy enhancement through enhanced degradation of aggregated proteins. Additionally, in a C57BL/6 mice model with MPTP-induced substantia nigra injuries, MYH inhibits both the loss of TH-positive neurons in the substantia nigra pars compacta (SNpc) and the reduction of the optical density of TH-IR fibers in the striatum (ST). CONCLUSIONS All of our results indicate that MYH treatment has neuroprotective effects that are partially mediated by autophagy enhancement. MYH may be a promising herbal formula for the prevention and treatment of neurodegenerative diseases, especially PD.

Induction of autophagy by dimethyl cardamonin is associated with proliferative arrest in human colorectal carcinoma HCT116 and LOVO cells

Dimethyl cardamonin (2′,4′‐dihydroxy‐6′‐methoxy‐3′,5′‐dimethylchalcone; DMC) is a naturally occurring chalcone, and it is the major compound isolated from the leaves of Syzygium samarangense (Blume) Merr. & L.M. Perry (Myrtaceae). Experiments were conducted to determine the effects of DMC on cell proliferation, cell‐cycle distribution, and programmed cell death in cultures of human colorectal carcinoma HCT116 and LOVO cells. Results showed that DMC inhibited HCT116 and LOVO cell proliferation and induced G2/M cell cycle arrest, which was associated with the conversion of microtubule associated protein light chain 3 (LC3)‐I–LC3‐II, an autophagosome marker, and the incorporation of monodansylcadaverine (MDC), a marker for the acidic compartment of autolysosomes or acidic vesicular organelles. The treatment of HCT116 and LOVO cells using a combination of DMC with an autophagy inhibitor, such as 3‐methyladenine (3‐MA), beclin 1 siRNA, or atg5 siRNA, suppressed the effect of DMC‐mediated anti‐proliferation. These results imply that DMC can suppress colorectal carcinoma HCT116 and LOVO cell proliferation through a G2/M phase cell‐cycle delay, and can induce autophagy, the hallmark of Type II programmed cell death (PCD). Taken together, our results suggest that DMC may be an effective chemotherapeutic agent for HCT116 and LOVO colorectal carcinoma cells. J. Cell. Biochem. 112: 2471–2479, 2011.

Coprismycins A and B, neuroprotective phenylpyridines from the dung beetle-associated bacterium, Streptomyces sp.

Two new phenylpyridines, named coprismycins A and B (1 and 2), and previously reported dipyridines (3-6) were isolated from a culture of Streptomyces sp. associated with the dung beetle, Copris tripartitus. The structures of the coprismycins (1 and 2) were elucidated by the analysis of 1D and 2D NMR spectra and mass, UV, and IR spectra. Coprismycins A-B (1 and 2) and dipyridines (5 and 6) displayed comparable neuroprotective effects against MPP(+) (1-methyl-4-phenylpyrimidium)-induced neurotoxicity in neuroblastoma SH-SY5Y cells.

Inhibition of A2780 Human Ovarian Carcinoma Cell Proliferation by a Rubus Component, Sanguiin H-6

The effects of a red raspberry component, sanguiin H-6 (SH-6), on the induction of apoptosis and the related signaling pathways in A2780 human ovarian carcinoma cells were investigated. SH-6 caused an antiproliferative effect and a severe morphological change resembling that of apoptotic cell death but no effect on the cancer cell cycle arrest. In addition, SH-6 induced an early apoptotic effect and activation of caspases as well as the cleavage of PARP, which is a hallmark of apoptosis. The early apoptotic percentages of A2780 cells exposed to 20 and 40 μM SH-6 were 35.39 and 41.76, respectively. Also, SH-6 caused the activation of mitogen-activated protein kinases (MAPKs), especially p38, and the increase of truncated p15/BID. These results in the present study suggest that the apoptosis of A2780 human ovarian carcinoma cells by SH-6 is mediated by the MAPK p38 and a caspase-8-dependent BID cleavage pathway.

Neuroprotective effect of modified Wu-Zi-Yan-Zong granule, a traditional Chinese herbal medicine, on CoCl2-induced PC12 cells

AIM OF THE STUDY To investigate the neuroprotective effect of aqueous extract of modified Wu-Zi-Yan-Zong granule (MWG), a traditional Chinese herbal medicine, against CoCl(2)-induced neurotoxicity in PC12 cells. MATERIALS AND METHODS Cell viability assay, apoptosis rate assay, ROS detection and mitochondrial membrane potential (MMP) assay were performed. In addition, cytochrome c, caspase-3, PARP and MAPKs were also detected by Western blotting. RESULTS MWG extract increased viability and suppresses early and middle/late stage apoptosis in a dose-dependent manner in CoCl(2)-induced PC12 cells. Moreover, MWG extract decreased the level of intracellular reactive oxygen species (ROS), increased MMP, regulated Bcl-2 family protein expression (Bcl-2 and Bcl-XL) and inhibited the release of cytochrome c from the mitochondria. In addition, MWG extract attenuated activation of caspase-3 and poly ADP-ribose polymerase (PARP) and inhibited the phosphorylation of ERK, c-Jun NH(2)-terminal kinase (JNK) and p38 MAPKs. CONCLUSIONS MWG extract exhibited significant neuroprotective effect on PC12 cells, and this effect may be associated with the suppression of ROS generation and inhibition of mitochondria-mediated caspase and MAPK signaling pathways.

Stercurensin inhibits nuclear factor-κB-dependent inflammatory signals through attenuation of TAK1–TAB1 complex formation

We identified a chalcone, 2′,4′‐dihydroxy‐6′‐methoxy‐3′‐methylchalcone (stercurensin), as an active compound isolated from the leaves of Syzygium samarangense. In the present study, the anti‐inflammatory effects and underlying mechanisms of stercurensin were examined using lipopolysaccharide (LPS)‐stimulated RAW264.7 cells and mice. To determine the effects of stercurensin in vitro, inducible nitric oxide synthase (iNOS) and cyclooxygenase‐2 (COX‐2) expression were analyzed by RT‐PCR and immunoblotting. Nuclear factor‐κB (NF‐κB) activation and its upstream signaling cascades were also investigated using a dual‐luciferase reporter assay, electrophoretic mobility shift assay, immunoblotting, immunofluorescence, and immunoprecipitation. To verify the effects of stercurensin in vivo, the mRNA expression levels of iNOS and COX‐2 were evaluated in isolated mouse peritoneal macrophages by quantitative real‐time PCR, and the production of tumor necrosis factor‐α (TNF‐α), interleukin‐6 (IL‐6), and IL‐1β were assessed in serum samples from mice using a Luminex system. Pretreatment with stercurensin reduced LPS‐induced iNOS and COX‐2 expression, thereby inhibiting nitric oxide (NO) and prostaglandin E2 production, respectively. In addition, an inhibitory effect of stercurensin on NF‐κB activation was shown by the recovery of LPS‐induced inhibitor of κB (I‐κB) degradation after blocking the transforming growth factor‐β‐activated kinase 1 (TAK1)/I‐κB kinase signaling pathway. In mouse models, stercurensin negatively regulated NF‐κB‐dependent pro‐inflammatory mediators and cytokines. These results demonstrate that stercurensin modulates NF‐κB‐dependent inflammatory pathways through the attenuation of TAK1–TAB1 complex formation. Our findings demonstrating the anti‐inflammatory effects of stercurensin in vitro and in vivo will aid in understanding the pharmacology and mode of action of stercurensin. J. Cell. Biochem. 112: 548–558, 2011.

Inhibitory effects of aurentiacin from Syzygium samarangense on lipopolysaccharide-induced inflammatory response in mouse macrophages

Aurentiacin is a chalcone isolated from Syzygium samarangense. In the present study, we examined the anti-inflammatory effects of aurentiacin in lipopolysaccharide (LPS)-stimulated mouse macrophages. Aurentiacin significantly inhibited LPS-induced nitric oxide (NO) production in RAW264.7 cells concomitantly with the suppression of inducible nitric oxide synthase (iNOS) expression. Aurentiacin also reduced the mRNA levels of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Electrophoretic mobility shift assays (EMSAs) and reporter gene assays indicated that DNA binding and transcriptional activities of nuclear factor-κB (NF-κB)/p65 were decreased by aurentiacin in LPS-stimulated RAW264.7 cells. Moreover, results from chromatin immunoprecipitation (ChIP) assays over the promoter region of the iNOS gene were in agreement with the EMSA results. Pretreatment with aurentiacin prevented the nuclear translocation of p65 by blocking the phosphorylation of I-κB kinase (IKK). Aurentiacin also attenuated the phosphorylation (Ser536) and acetylation (Lys310) of p65 and phosphorylation of MAPKs. In an inflammatory animal model, the intraperitoneal (i.p.) injection of aurentiacin suppressed the release of pro-inflammatory cytokines. Moreover, the level of iNOS protein ex vivo was decreased by aurentiacin similar to the result in vitro. Taken together, these results suggest that aurentiacin shows anti-inflammatory activity related to the inhibition of NF-κB activation.

Targeting CPT1A enhances metabolic therapy in human melanoma cells with the BRAF V600E mutation

Cancer cells are characterized by altered metabolic processes. Recent evidence of metabolic alterations has indicated that the fatty acid oxidation (FAO) pathway is used as a carbon source for anabolic processes in some tumors, thus making this pathway a potential target for therapy. The carnitine palmitoyltransferase (CPT; EC 2.3.1.21) enzyme transfers long-chain fatty acids from the cytosol to the mitochondrial matrix for β-oxidation. Because carnitine palmitoyl transferase 1a (CPT1a) is the rate-limiting enzyme for FAO, the authors evaluated the effects of CPT1A knock-down in BRAF V600E melanoma cell lines. The results showed that knock-down of CPT1A inhibited FAO and that CPT1A is critical for malignant V600E melanoma cells, particularly BRAF V600E melanoma cells. The proliferation and tumorigenesis in V600E melanoma were decrease after CPT1A knockdown. These results suggest that therapy for BRAF V600E melanoma can include targeting metabolic alterations. CPT1A is more important for lipid synthesis in V600E mutant melanoma cells than in wild-type BRAF melanoma cells.