• Applied Biological Chemistry
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• v.51 no.2
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• pp.129-135
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• 2008

Berberine is an isoquinoline alkaloid used in traditional Chinese medicine and has been isolated from a variety of plants, such as Coptis chinensis and Phellodendron amurense. It has a wide spectrum of clinical applications such as in anti-tumor, anti-microbial, and anti-inflammatory activities. However, it is still unknown that berberine related with reactive oxygen species (ROS)-mediated apoptosis pathway in human hepatoma HepG2 cells. In the present study, we are examined the molecular mechanism of ROS- and p38 MAP kinase-mediated apoptosis by berberine in HepG2 cells. Berberine increased cytotoxicity effects by time- and does-dependent manner. $LD_{50}$ was detected 50 ${\mu}M$ at 48h of exposure to berberine. Nuclei cleavage and apoptotic DNA fragmentation were observed in cells treated with 50 ${\mu}M$ of berberine for 48h. Moreover, berberine induced the activating of caspase-3, p53, p38 and Bax expression, whereas the expression of anti-apoptotic signaling pathways, Bcl-2, was decreased. Additionally, berberine-treated cells had an increased level of generation of ROS and nitric oxide (NO). These results indicated that berberine induces apoptosis of HepG2 cells may be mediated oxidative injury acts as an early and upstream change, triggers mitochondrial dysfunction, Bcl-2 and Bax modulation, p38 and p53 activation, caspase-3 activation, and consequent leading to apoptosis.

• Journal of the Korean Society of Food Science and Nutrition
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• v.37 no.5
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• pp.555-560
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• 2008

Amino acid transporters are essential for the growth and proliferation in all living cells. Among the amino acid transporters, the system L amino acid transporters are the major nutrient transport system responsible for the $Na^+$-independent transport of neutral amino acids including several essential amino acids. The L-type amino acid transporter 1 (LAT1), an isoform of system L amino acid transporter, is highly expressed in cancer cells to support their continuous growth and proliferation. 2-Aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH) is a model compound for the study of amino acid transporter as a system L selective inhibitor. We have examined the effect and mechanism of BCH on cell growth suppression in HEp2 human head and neck squamous cell carcinoma. The BCH inhibited the L-leucine transport in a concentration-dependent manner with a $IC_{50}$ value of $51.2{\pm}3.8{\mu}M$ in HEp2 cells. The growth of HEp2 cells was inhibited by BCH in the timeand concentration-dependent manners. The formation of DNA ladder was not observed with BCH treatment in the cells. Furthermore, the proteolytic processing of caspase-3 and caspase-7 in the cells were not detected by BCH treatment. These results suggest that the BCH inhibits the growth of HEp2 human head and neck squamous cell carcinoma through the intracellular depletion of neutral amino acids for cell growth without apoptotic processing.

• Herbal Formula Science
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• v.25 no.1
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• pp.51-68
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• 2017

Objectives : Oxidative stress due to excessive accumulation of reactive oxygen species (ROS) is one of the risk factors for the development of several chronic diseases, including neurodegenerative diseases. Methods : In the present study, we investigated the protective effects of cheongnoemyeongsin-hwan (CNMSH) against oxidative stress‑induced cellular damage and elucidated the underlying mechanisms in neuronal-derived SH-SY5Y cells. Results : Our results revealed that treatment with CNMSH prior to hydrogen peroxide (H2O2) exposure significantly increased the SH-SY5Y cell viability, indicating that the exposure of the SH-SY5Y cells to CNMSH conferred a protective effect against oxidative stress. CNMSH also effectively attenuated H2O2‑induced comet tail formation, and decreased the phosphorylation levels of the histone ${\gamma}H2AX$, as well as the number of apoptotic bodies and Annexin V‑positive cells. In addition, CNMSH exhibited scavenging activity against intracellular ROS generation and restored the mitochondria membrane potential (MMP) loss that were induced by H2O2, suggesting that CNMSH prevents H2O2‑induced DNA damage and cell apoptosis. Moreover, H2O2 enhanced the cleavage of caspase-3 and degradation of poly (ADP-ribose)-polymerase, a typical substrate protein of activated caspase-3, as well as DNA fragmentation; however, these events were almost totally reversed by pretreatment with CNMSH. Furthermore, CNMSH increased the levels of heme oxygenase-1 (HO-1), which is a potent antioxidant enzyme, associated with the induction of nuclear factor-erythroid 2-related factor 2 (Nrf2). According to our data, CNMSH is able to protect SH-SY5Y cells from H2O2-induced apoptosis throughout blocking cellular damage related to oxidative stress through a mechanism that would affect ROS elimination and activating Nrf2/HO-1 signaling pathway. Conclusions : Therefore, we believed that CNMSH may potentially serve as an agent for the treatment and prevention of neurodegenerative diseases caused by oxidative stress.

• Journal of the Korean Society of Food Science and Nutrition
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• v.45 no.6
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• pp.911-917
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• 2016

Lespedeza cuneata G. Don is an edible perennial herb used in traditional Korean medicine. We investigated the anti-proliferative properties and mechanism of L. cuneata extract. The ethanolic extract of L. cuneata dose-and time-dependently inhibited human colorectal cancer cell proliferation. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay was used to test the effect of the extract on proliferation of HT-29 colorectal cancer cells. The extract inhibited HT-29 cell proliferation with an $IC_{50}$ value of $554.26{\pm}8.81{\mu}g/mL$. L. cuneata extract suppressed production of pro-inflammatory cytokines interleukin-6 and tumor necrosis $factor-{\alpha}$. Apoptosis was evaluated by analysis of DNA fragmentation, poly(ADP-ribose) polymerase cleavage, caspase-3 activity, and protein expression of pro-apoptotic (Bax) and anti-apoptotic (Bcl-2). Our results demonstrated that the extract induced DNA fragmentation and characteristic morphological changes associated with apoptosis in HT-29 colorectal cancer cells. The extract also time- and dose-dependently up-regulated expression of the Bax and down-regulated expression of the Bcl-2. Furthermore, the extract dose- and time-dependently enhanced caspase-3 activity. Our findings provide evidence that L. cuneata extract may mediate its anti-proliferative effect via modulation of apoptosis.

• Korean Journal of Animal Reproduction
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• v.24 no.4
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• pp.439-449
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• 2000

This study was to investigate the expression of transgene after co-injection of spermatozoon and EGFP gene into mature oocytes in cattle. From frozen semen, spermatozoa were treated by DTT with 0.03% Tween-20, freezing and thawing or 0.02% Triton X-100 to disrupt their plasma membranes. The sperm injected oocytes were co-cultured with bovine oviduct epithelial cells in CRlaa, and expression of EGFP in embryos were observbed under epifluorescent microscope. Two pronuclei were formed in oocytes injected with sperm treated by DTT (44.6%), DTT-Tween-20 (48.4%), DTT-freezing (44.4%) and DTT-Triton X-100 (42.9%). Cleavage and blastocyst formation rates of bovine oocytes which injected with sperm treated by DTT, DTT-Tween-20, DTT-freezing, and DTT-Triton X-100 were 49.1, 58.5, 43.9, and 48.4% and 10.2, 13.0, 6.8, and 6.5%, respectively. Although the most of embryos were showing mosaicism, embryos expressing EGFP gene were observed in all treated groups. Therefore, these results indicate that membrane disrupted sperm could interact with exogenous DNA, and that this procedure may be useful to introduce foreign gene into bovine oocytes.

• Horticultural Science & Technology
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• v.34 no.6
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• pp.912-923
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• 2016

Watermelon production is often limited by powdery mildew in areas with a large daily temperature range. Development of resistant watermelon cultivars can protect against powdery mildew; however, little is known about the characteristics of its causal agents. Here, we identified the genus and race of a causal pathogen of powdery mildew in Ansung province of South Korea, and developed molecular markers for the generation of resistant watermelon cultivars. The causal pathogen was determined to be Podosphaera xanthii based on multiple sequence alignments of internal transcribed spacers (ITS) of rDNA. The physiological race was identified as 1W, and the Ansung isolate was named P. xanthii 1W-AN. Following inoculation with the identified P. xanthii 1W-AN, we found inheritance of the resistant gene fitting a single dominant Mendelian model in a segregated population ('SBA' ${\times}$ PI 254744). To develop molecular markers linked to fungus-resistant loci, random amplified polymorphic DNA (RAPD) was accomplished between DNA pooled from eight near-isogenic lines (NILs; $BC_4F_6$), originated from PI 254744 and susceptible 'SBB' watermelon. After sequencing bands from RAPD were identified in all eight NILs and PI254744, 42 sequence-characterized amplifiedregion (SCAR) markers were developed. Overall, 107 $F_2$ plants derived from $BC_4F_6$ NIL-1 ${\times}$ 'SBB' were tested, and one SCAR marker was selected. Sequence comparison between the SCAR marker and the reference watermelon genome identified three Nco I restriction enzyme sites harboring a single nucleotide polymorphism, and codominant cleavage-amplified polymorphic site markers were subsequently developed. A CAPS marker was converted to a high-resolution melt (HRM) marker, which can discriminate C/T SNP (254PMR-HRM3). The 254PMR-HRM3 marker was evaluated in 138 $F_{2:3}$ plants of a segregating population ('SBA' ${\times}$ PI254744) and was presumed to be 4.3 cM from the resistance locus. These results could ensure P. xanthii 1W-AN resistance in watermelon germplasm and aid watermelon cultivar development in marker-assist breeding programs.

• Journal of Ginseng Research
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• v.28 no.1
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• pp.39-44
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• 2004

The present study is to determine whether the Red-ginseng extract has a proliferative or cytotoxic effect on the human neuronal stem cells(hNSCs). The hNSCs were grown and incubated with different doses of Red-ginseng extract. We tested the proliferative or cytotoxic effects by MTT and FACS analysis. Cell viability cell cycle analysis, DNA fragmentation, and bax or PARP expressions were evaluated. The hNSCs showed a proliferafe trend with its peak concentration at 0.3 $\mu\textrm{g}$/$m\ell$. Beyond this point, higher doses decreased viabilities and showed a cytotoxic effect at 10 $\mu\textrm{g}$/$m\ell$. There was a tendency of increased S and G2/M phases during cell proliferation. In a cytotoxic condition, decreased S phase and increased G0/G1 phases were noted, suggesting cell cycle arrest. The cytotoxic effect was associated with increase DNA fragmentation in a dose-dependent manner, However PARP cleavage or bax expression was not detected. Our results suggest that Red-ginseng extract has dual effects, the cell proliferative or cytotoxic effect, on hNSCs in vitro with dose-dependent manner.

• Journal of Life Science
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• v.19 no.8
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• pp.1047-1054
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• 2009

Oral squamous carcinoma (OSC) cells present resistance to chemotherapeutic agents-mediated apoptosis in the late stages of malignancy. Advances in the understanding of bacterial toxins have produced new strategies for the treatment of cancers. It was demonstrated here that Pseudomonas aeruginosa exotoxin A (PEA) significantly decreased the viability of chemoresistant YD-9 cells in the apoptosis mechanism. Apoptotic manifestations were evident through changes in nuclear morphology and generation of DNA fragmentation. PEA treatment induced caspase-3, -6 and -9 cleavage, and activation. These events preceded proteolysis of the caspase substrates poly (ADP-ribose) polymerase (PARP), DNA fragmentation factor 45 (DFF45), and lamin A in YD-9 cells. The reduction of mitochondrial membrane potential, release of cytochrome c and SmacjDlABLO from mitochondria to cytosol, andtranslocation of AlF into nucleus were shown. While p53, p21 and $14-3-3{\gamma}$ were upregulated, cyclin Band cdc2 were downregulated by PEA treatment. Taken together, PEA induces apoptosis in chemoresistant YD-9 cells via activation of caspases, mitochondrial events and regulation of cell cycle genes.

• Korean Journal of Ecology and Environment
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• v.36 no.2 s.103
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• pp.108-116
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• 2003

To investigate bacteria with algal Iytic activities against Anabaena cylindrica when water blooming occurs and to study enzyme profiles of alga-Iytic bacteria, various bacterial strains were isolated from surface waters and sediments in eutrophic lakes or reservoirs in Korea. Abacterial strain AK-07 was characterized and identified as Acinetobacter johnsonii based on its16S rDNA base sequence. When AK-07 was co-cultivated with A. cylindrica, bacterial cells propagated to $8\;{\times}\;10^8$ cfu $ml^{-1}$ and Iyses algal cells. However, culture filtrates of AK-07 did not exhibit algal Iytic activities. That suggesting the enzymes on the surfaces of the bacterium might be effective algal Iytic agents to cause Iyses of cells. Acinetobacter johnsonii AK-07 exhibited high degradation activities against A. cylindrica, and formed alginase, caseinase, lipase, fucodian hydrolase, and laminarinase. Moreover, glycosidases for example ${\beta}$-galatosidase, ${\beta}$-glucosidase, ${\beta}$-glucosaminidase, and ${\beta}$-xylosidase, which hydrolyzed ${\beta}$-0-glycosidic bonds, were found in cell-free extracts of A. johnsonii AK-07. Other glycosidase such as ${\alpha}$-galctosidases, ${\alpha}$-N-Ac-galctosidases, ${\alpha}$-mannosidases, and ${\alpha}$- L-fuco-sidases, which cleavage ${\alpha}$-0-glycosidic bondsare not detected. In the results, enzyme systemsof A. johnsonii AK-07 were very complex to do-grade cell walls of cyanobacteria. The polysaccharides or peptidoglycans of A. cylindrica maybe hydrolyzed and metabolized to a range of easily utilizable monosaccharides or other low molecular weight organic substances by strain AK-07 of A. johnsonii.

• The Korea Journal of Herbology
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• v.29 no.2
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• pp.1-6
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• 2014

Objectives : Reactive oxygen species (ROS) are involved in a wide spectrum of diseases including chronic inflammation and cancer. In this study, we investigated the antioxidant activities and anti-inflammatory effects of the extracts from the herbal teas such as Lonicera japonica Thunberg (L. japonica), Chrysanthemum morifolium Ramat (C. morifolium), Mentha arvensis L. (M. arvensis), and P.rhizoma. Methods : Anti-oxidant activity was evaluated using DPPH radical scavenging assay and $Fe^{2+}$ chelating assay. And DNA cleavage assay was performed to evaluate an anti-oxidative effect. Anti-inflammatory effect was performed using NO generation assay and western blot in LPS-stimulated RAW264.7 cell line. Results : L. japonica scavenged DPPH radical by 9.8% at 12.5 ${\mu}g/ml$, 24.8% at 25 ${\mu}g/ml$, 34.3% at 50 ${\mu}g/ml$, 61.1% at 100 ${\mu}g/ml$ and 75.8% at 200 ${\mu}g/ml$, respectively. In addition, C. morifolium and M. arvensis removed DPPH radical by 15.6% and 10.4% at 12.5 ${\mu}g/ml$, 34.8% and 22.8% at 25 ${\mu}g/ml$, 66.9% and 43.3% at 50 ${\mu}g/ml$, 87.4% and 69.1% at 100 ${\mu}g/ml$, and 92.1% and 73.2% at 200 ${\mu}g/ml$, respectively. However, P. rhizoma did not affect on DPPH radical scavenging. The $Fe^{2+}$ chelating activity was highest in L. japonica, but lowest in P. rhizoma among the herbal teas. In addition, the extracts from L. japonica, C. morifolium and M. arvensis inhibited oxidative DNA damage via its anti-oxidant activity. In anti-inflammatory effect, the extracts from C. morifolium inhibited NO production. In addition, it suppressed the $NF-{\kappa}B$ signaling pathway in LPS-stimulated RAW 264.7 cells. Conclusions : Together, this study indicates that L. japonica, M. arvensis and C. morifolium possess the protective effect against the oxidative DNA damage. Furthermore, C. morifolium exerts an anti-inflammatory effect.