Young-Sool Hah

Acetoacetate protects neuronal cells from oxidative glutamate toxicity

Glutamate cytotoxicity contributes to neuronal degeneration in many central nervous system (CNS) diseases, such as epilepsy and ischemia. We previously reported that a high‐fat and low‐carbohydrate diet, the ketogenic diet (KD), protects against kainic acid‐induced hippocampal cell death in mice. We hypothesized based on these findings that ketosis resulting from KD might inhibit glutamate cytotoxicity, resulting in inhibition of hippocampal neuronal cell death. Therefore, we investigated the role of ketone bodies [acetoacetate (AA) and β‐hydroxybutyrate (β‐OHB)] both in a mouse hippocampal cell line (HT22) and in rat primary hippocampal neurons. As a result, we found that pretreatment with 5 mM lithium AA and 4 mM Na β‐OHB protected the HT22 hippocampal cell line and primary hippocampal neuronal culture against 5 mM glutamate toxicity and that up to 2 hr of pretreatment with 5 mM AA had a protective effect against 5 mM glutamate toxicity in the HT22 cell line. Pretreatment with 5 mM AA decreased ROS production of HT22 cell line at 2 and 8 hr exposure of glutamate, and it decreased the appearance of annexin V‐positive HT22 cells, which are indicative of an early stage of apoptosis, and propidium iodide‐positive HT22 cells, which are indicative of necrosis.

A20 suppresses inflammatory responses and bone destruction in human fibroblast‐like synoviocytes and in mice with collagen‐induced arthritis

OBJECTIVE Nuclear factor-kappaB (NF-kappaB) has been implicated as a therapeutic target for the treatment of rheumatoid arthritis (RA). The purpose of this study was to determine whether A20, a universal inhibitor of NF-kappaB, might have antiarthritic effects. METHODS An adenovirus containing A20 complementary DNA (AdA20) was used to deliver A20 to human rheumatoid fibroblast-like synoviocytes (FLS) in vitro as well as to mice with collagen-induced arthritis (CIA) in vivo via intraarticular injection into the ankle joints bilaterally. RESULTS In vitro experiments demonstrated that AdA20 suppressed NF-kappaB activation, chemokine production, and matrix metalloproteinase secretion induced by tumor necrosis factor alpha in FLS. Mice with CIA that were treated with AdA20 had a lower cumulative disease incidence and severity of arthritis, based on hind paw thickness, radiologic and histopathologic findings, and inflammatory cytokine levels, than did control virus-injected mice. The protective effects of AdA20 were mediated by the inhibition of the NF-kappaB signaling pathway. The severity of arthritis was also significantly decreased in the untreated front paws, indicating a beneficial systemic effect of local suppression of NF-kappaB. Surprisingly, mice treated with AdA20 after the onset of CIA had significantly decreased arthritis severity from the onset of clinical signs to the end of the study. CONCLUSION These results suggest that using A20 to block the NF-kappaB pathway in rheumatoid joints reduces both the inflammatory response and the tissue destruction. The development of an immunoregulatory strategy based on A20 may therefore have therapeutic potential in the treatment of RA.

In vitro and in vivo osteogenesis of human mesenchymal stem cells derived from skin, bone marrow and dental follicle tissues

The present study evaluated the human mesenchymal stem cells (hMSCs) isolated from skin (hSMSC), bone marrow (hBMSC) and dental follicle (hDFMSC) tissues on their in vitro and in vivo osteogenic potential using demineralized bone matrix (DBM) and fibrin glue scaffold. Cells originated from three distinct tissues showed positive expressions of CD44, CD73, CD90, CD105 and vimentin, and differentiation ability into osteocytes, adipocytes and chondrocytes. hMSCs from all tissues co-cultured with a mixed DBM and fibrin glue scaffold in non-osteogenic induction media were positively stained by von Kossa and expressed osteoblast-related genes, such as osteocalcin (OC), osteonectin (ON), runt-related transcription factor 2 (Runx2) and osterix. For in vivo osteogenic evaluation, PKH26 labeled hMSCs were implanted into the subcutaneous spaces of athymic mice with a mixed scaffold. At 4 weeks of implantation, PKH26 labeled cells were detected in all hMSC-implanted groups. Bone formation with OC expression and radio-opacity intensity were observed around DBM scaffold in all hMSC-implanted groups. Interestingly, hDFMSCs-implanted group showed the highest OC expression and calcium content. These findings demonstrated that hDFMSCs could be a potential alternative autologous cell source for bone tissue engineering.

Cathepsin D inhibits oxidative stress-induced cell death via activation of autophagy in cancer cells

Cathepsin D (CatD), a lysosomal aspartic protease, plays an essential role in tumor progression and apoptosis. However, the function of CatD in cell death is not yet fully understood. In this study, we identified CatD as one of up-regulated proteins in human malignant glioblastoma M059J cells that lack the catalytic subunit of DNA-PK compared with its isogenic M059K cells with normal DNA-PK activity. M059J cells were relatively more resistant to genotoxic stress than M059K cells. Overexpression of wild-type CatD but not catalytically inactive mutant CatD (D295N) inhibited H(2)O(2)-induced cell death in HeLa cells. Furthermore, knockdown of CatD expression abolished anti-apoptotic effect by CatD in the presence of H(2)O(2). Interestingly, high expression of CatD in HeLa cells significantly activated autophagy: increase of acidic autophagic vacuoles, LC3-II formation, and GFP-LC3 puncta. These results suggest that CatD can function as an anti-apoptotic mediator by inducing autophagy under cellular stress. In conclusion, inhibition of autophagy could be a novel strategy for the adjuvant chemotherapy of CatD-expressing cancers.

Polyphenolic extract isolated from Korean Lonicera japonica Thunb. induce G2/M cell cycle arrest and apoptosis in HepG2 cells: involvements of PI3K/Akt and MAPKs.

Lonicera japonica Thunb. (L. japonica T.) has been used in Korean traditional medicine for long time because of its anti-cancer and hepatic protective effect. In this study, we investigated polyphenolic extract in L. japonica T. using high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) and its anti-cancer effect on hepatocarcinoma cells. Human HepG2 cell line was treated with various concentrations of polyphenolic extract. Apoptosis was detective by cell morphology, cell cycle analysis and immunoblot analysis. Polyphenolic extract inhibited cell proliferation at 48h in a dose-dependent manner. Polyphenolic extract affected HepG2 cell viability by inhibiting cell cycle progression at the G2/M transition and inducing apoptosis. Polyphenolic extract also decreased the expression of CDK1, CDC25C, cyclin B1, pro-caspases-3 and -9 and poly ADP ribose polymerase, and affected the levels of mitochondrial apoptotic-related proteins. The phosphorylation of extracellular signal-related kinase ½ (ERK 1/2), c-Jun N-terminal kinase (JNK), and p-38 mitogen-activated protein kinases (MAPKs) were increased in HepG2 cells treated with polyphenolic extract, whereas Akt was dephosphorylated. These results indicate that inhibition of PI3K/Akt and activation of MAPKs are pivotal in G2/M cell cycle arrest and apoptosis of human hepatocarcinoma cells mediated by polyphenolic extract.

Flavonoids Isolated from Korea Citrus aurantium L. Induce G2/M Phase Arrest and Apoptosis in Human Gastric Cancer AGS Cells

Aim of the Study. Citrus species is used in traditional medicine as medicinal herb in several Asian countries including Korea. Flavonioids became known as various properties, such as anti-oxidants, anti-inflammation and anti-cancer, and so forth. The present study, the anti-cancer effect of flavonioids isolated from Citrus aurantium L. in human gastric cancer AGS cells has been investigated. Materials and Methods. The anti-proliferative activity was assayed using MTT assay. Cell cycle analysis was done using flow cytometry and apoptosis detection was done using by hoechst fluorescent staining and Annexin V-propidium iodide double staining. Western blot was used to detect the expression of protein related with cell cycle and apoptosis. Results. Flavonoids isolated from Citrus aurantium L. have the effect of anti proliferation on AGS cells with IC50 value of 99 μg/mL. Flavonoids inhibited cell cycle progression in the G2/M phase and decrease expression level of cyclin B1, cdc 2, cdc 25c. Flavonoids induced apoptosis through activate caspase and inactivate PARP. Conclusions. Flavonoids isolated from Citrus aurantium L. induced G2/M phase arrest through the modulation of cell cycle related proteins and apoptosis through activation caspase. These finding suggest flavonoids isolated from Citrus aurantium L. were useful agent for the chemoprevention of gastric cancer.

Propofol protects the autophagic cell death induced by the ischemia/reperfusion injury in rats

Autophagy has been implicated in cardiac cell death during ischemia/reperfusion (I/R). In this study we investigated how propofol, an antioxidant widely used for anesthesia, affects the autophagic cell death induced by the myocardial I/R injury. The infarction size in the myocardium was dramatically reduced in rats treated with propofol during I/R compared with untreated rats. A large number of autophagic vacuoles were observed in the cardiomyocytes of I/R-injured rats but rarely in I/R-injured rats treated with propofol. While LC3-II formation, an autophagy marker, was up-regulated in the I/R-injured myocardium, it was significantly down-regulated in the myocardial tissues of I/R-injured and propofol-treated rats. Moreover, propofol inhibited the I/R-induced expression of Beclin-1, and it accelerated phosphorylation of mTOR during I/R and Beclin-1/Bcl-2 interaction in cells, which indicates that it facilitates the inhibitory pathway of autophagy. These data suggest that propofol protects the autophagic cell death induced by the myocardial I/R injury.

Induction of Apoptosis and Inhibition of Invasion in Human Hepatoma Cells by Anthocyanins from Meoru

Anthocyanins belong to a class of flavonoids exhibiting antioxidant and anti‐inflammatory actions as well as a variety of chemotherapeutic effects. However, little is known about the cellular and molecular mechanism of anticancer activity. In this study, we investigated if the anthocyanins (delphinidin‐3,5‐diglucoside: cyanidin‐3,5‐diglucoside: petunidin‐3,5‐diglucoside: delphinidin‐3‐glucoside: malvdin‐3,5‐diglucoside: peonidin‐3,5‐diglucoside: cyanidin‐3‐glucoside: petunidin‐3‐glucoside: peonidin‐3‐ glucoside: malvidin‐3‐ glucoside = 27: 63: 8.27: 1:2.21: 6.7: 1.25: 5.72: 1.25) isolated from meoru (Vitis coignetiae Pulliat) exerted antiproliferative and anti‐invasive and apoptotic effects on human hepatoma Hep3B cells. It was found that the anthocyanins could inhibit cell growth by 75% at the concentration of 400 μg/mL for 48 h. Flow cytometric analysis showed that the anthocyanins increased the amount of DNA fragments (sub‐G1 fraction) in a dose‐dependent manner, which is closely related to mitochondrial dysfunction and reduction in antiapoptotic proteins (Bcl‐2, xIAP, cIAP‐1, and cIAP‐2). The anthocyanins also significantly inhibited the migration and invasion of Hep3B cells through a matrigel‐coated chamber. Taken together this study indicates that the anthocyanins from meoru have antiproliferative and anti‐invasive effects and may induce apoptosis through the activation of the mitochondrial pathway and inhibition of antiapoptotic proteins. This study provides evidence that the anthocyanins isolated from meoru might be useful in the treatment of human hepatitis B‐associated hepatoma.

Antiapoptotic effects of anthocyanins on rotator cuff tenofibroblasts.

Degeneration of the rotator cuff tendon, which involves apoptosis of the tenofibroblasts, is one of the most common shoulder problems that can lead eventually to a full‐thickness rotator cuff tendon tear. The current authors evaluated both the ability of anthocyanins, which are powerful antioxidants, to reduce apoptosis in oxidation‐stressed rotator cuff tenofibroblasts, and the molecular mechanism for this antiapoptotic action. Anthocyanins demonstrated a dose‐dependent ability to inhibit H2O2‐induced apoptosis in cultured tenofibroblasts, as assessed by MTT assay and FACS analysis. H2O2 increased the phosphorylation of extracellular regulated kinase1/2 (ERK1/2) and of c‐Jun N‐terminal kinase (JNK) and the production of reactive oxygen species (ROS). In contrast, treatment with anthocyanins decreased this activation of ERK1/2 and JNK, as confirmed by Western blot analysis, and reduced the production of ROS, as verified by fluorescent microscopic and FACS analyses. These findings suggest that anthocyanins, by suppressing JNK, ERK1/2, and intracellular ROS production, have a concentration‐dependent antiapoptotic effect on rotator cuff tenofibroblasts exposed to an oxidative stressor, and may have therapeutic potential.

Cytotoxic Effects of Ropivacaine, Bupivacaine, and Lidocaine on Rotator Cuff Tenofibroblasts

Background: Concern has recently arisen over the safety of local anesthetics used on human tissues. Hypothesis: Aminoamide local anesthetics have cytotoxic effects on human rotator cuff tenofibroblasts. Study Design: Controlled laboratory study. Methods: Cultured human rotator cuff tenofibroblasts were divided into control, phosphate buffered saline (PBS), and local anesthetic study groups; the PBS study group was further subdivided by pH level (pH 7.4, 6.0, and 4.4). The 6 local anesthetic subgroups (0.2% and 0.75% ropivacaine, 0.25% and 0.5% bupivacaine, and 1% and 2% lidocaine) were also studied at 10% dilutions of their original concentrations. Exposure times were 5, 10, 20, 40, or 60 minutes for the higher concentrations and 2, 6, 12, 24, 48, or 72 hours for the lower concentrations. Cell viability was evaluated through live, apoptotic, and necrotic cell rates using the annexin V–propidium iodide double-staining method. Intracellular reactive oxygen species (ROS) and the activity of mitogen-activated protein kinases (MAPKs) and caspase-3/7 were investigated. Results: The control and PBS groups showed no significant differences in cell viability (P > .999). In the local anesthetic study groups, cell viability decreased significantly with increases in anesthetic concentrations (P < .001) and exposure times (P < .001), with the exception of the lidocaine subgroups, where this effect was masked by the very high cytotoxicity of even low concentrations. Among the studied local anesthetic subgroups, 0.2% ropivacaine was the least toxic. The levels of intracellular ROS of each local anesthetic subgroup also increased significantly (P < .05). The studied local anesthetics showed increases in the phosphorylation of extracellular signal–regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38 as well as in levels of caspase-3/7 activity (P < .001). Conclusion: The cytotoxicity of the anesthetics studied to tenofibroblasts is dependent on exposure time and concentration. Of the evaluated anesthetics, ropivacaine is the least toxic in the clinically used concentration. The studied anesthetics induce tenofibroblast cell death, mediated by the increased production of ROS, by the increased activation of ERK1/2, JNK, and p38 and by the activation of caspase-3/7. Clinical Relevance: This study identified the cytotoxic mechanisms of aminoamide local anesthetics acting on rotator cuff tenofibroblasts. The greatest margin of safety was found in lower anesthetic concentrations in general and more specifically in the use of ropivacaine.