• Journal of Physiology & Pathology in Korean Medicine
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• v.18 no.4
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• pp.1055-1060
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• 2004

Apoptosis is the ability of cells to self-destruct by the activation of an intrinsic cellular suicide program when the cells are no longer needed or when they are seriously damaged. Morphologically, apoptosis is characterized by the appearance of membrane blebbing, cell shrinkage, chromatin condensation, DNA cleavage, and the fragmentation of the cell membrane-bound apoptotic bodies. Siegesbeckia glabrescens Makino (Siegesbeckiae Herba, SG) has been widely used as treatments for arthritis, and fever, as well as detoxification properties. The present studies were undertaken to evaluate if SG has an anti-apoptotic property. Cell viability was measured by XTT and tryphan blue stain. Morphological characteristic of human aortic smooth muscle cells(HASMC) were visualized with a phase-contrast microscope. SG significantly reduced HASMC, but not human umbilical vein endothelial cell(HUVEC), viability in a dose-dependent manner. Confluent untreated cells at 24hrs showed normal morphology, flat with a uniform polygonal shape. SG-treated cells (0.5㎎/㎖) at 24hrs showed apoptotic morphology. Cells became irregular with elongated lamellipodia, and exhibited condensed chromatin in nuclei with occasional endoucleation. There was an increase in the number of apoptotic cells rounding-up and being detached from the substrate. TUNEL staining of SG-treated cells showed dark-brown stains in nuclei and cytosol. Caspases are central components of the machinery responsible for apoptosis and are generally divided into two categories; the initiator caspases, which include caspases-2,-8,-9, and -10, and the effector caspases, which include caspases-3,-6, and -7. SG decreased anti-caspase-3 protein expression, which means activation of caspases-3 activity. It has been reported that there is a link between NO formation and apoptosis. NO production was accelerated by SG treatment in HASMC. L-NNA, NOS inhibitor, inhibited SG-induced apoptosis. These results, therefore, indicated that both caspases-3 and NO production are involved in apoptosis in smooth muscle cells. According to these results, SG may have a potential effect in the treatment of hypertensive atherosclerosis.

• Journal of Life Science
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• v.17 no.9 s.89
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• pp.1182-1190
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• 2007

The gene encoding for isoamylase of the Pectobacterium carotovorum subsp. carotovorum (Pcc) LY34 was cloned and expressed into Escherichia coli $DH5{\alpha}$. Isoamylase catalyzes the hydrolysis of ${\alpha}-1,6-glycosidic$ linkages specifically in amylopectin, glycogen, and derived oligosaccharides, while the enzyme did not hydrolyze ${\alpha}-1,4-glycosidic$ linkages of amylose. The isoamylase gene (glgX) had an open reading frame of 1,977 bp encoding 658 amino acid residues with a calculated molecular weight of 74,188 Da. The molecular weight of the enzyme was also estimated to be 74 kDa by activity staining of a SDS-PA gel. The mature GlgX had a calculated pI of 4.91. Isoamylase from Pcc LY34 had 70% amino acid identity with isoamylase from Pectobacterium chrysanthemi and contained the four regions conserved among all amylolytic enzymes. The isoamylase was optimally active at pH 7.0 and $40^{\circ}C$. GlgX was $Ca^{2+}-dependent$. The changes of Asp-335, Glu-370, and Asp-442 into Ala, respectively, using site-directed mutagenesis techniques showed that three residues are essential to isolamyalse (GlgX) activity. The sequences around those residues were highly conserved in isoamylase of different origins and GlgX of the glg operon in glycongen biosynthesis.

• Journal of the Korean Society of Food Science and Nutrition
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• v.38 no.8
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• pp.1003-1007
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• 2009

To develop Allium tuberosum L. as a cancer preventive food material, thiosulfinates and biological active components were isolated from Allium tuberosum L. and the apoptotic effects of thiosulfinates in human cancer cells were examined. Thiosulfinates decreased viable cell numbers in dose- and time-dependent manners. Thiosulfinates at the 20 $\mu g$/mL concentration inhibited more than 60% cell proliferation in HepG2 and A549 human cancer cells, respectively. Also the morphology of cells treated with thiosulfinates of 30 $\mu g$/mL concentration was distorted with shrunken cell mass while the cell number was lower than that of control cells. The $IC_{50}$ values in the HepG2 cells were higher than those of the A549 cells. Thiosulfinates at the 30 $\mu g$/mL concentration showed the formation of apoptotic bodies and a nuclear condensation, and an increase in the cell populations of the sub-G1 phase in the HepG2 cells. These results indicate that thiosulfinates from Allium tuberosum L. inhibited cell proliferation in HepG2 via apoptosis.

• BMB Reports
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• v.38 no.6
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• pp.755-762
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• 2005

Epstein-Barr virus (EBV) infects more than 90% of the world's population and has a potential oncogenic nature. A histone deacetylase (HDAC) inhibitor, trichostatin A (TSA), has shown potential ability in cancer chemoprevention and treatment, but its effect on EBV-infected Akata cells has not been examined. This study investigated the effect of TSA on the proliferation and apoptosis of the cells. TSA inhibited cell growth and induced cytotoxicity in the EBV infected Akata cells. TSA treatment sensitively induced apoptosis in the cell, which was demonstrated by the increased number of positively stained cells in the TUNEL assay, the migration of many cells to the sub-$G_0/G_1$ phase in flow cytometric analysis, and the ladder formation of genomic DNA. Western blot analysis showed that caspase-dependent pathways are involved in the TSA-induced apoptosis of EBV-infected Akata cells. Overall, this study shows that EBV-infected B lymphomas are quite sensitive to TSA-provoked apoptosis.

• Journal of Microbiology and Biotechnology
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• v.30 no.2
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• pp.279-286
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• 2020

A novel compound named 'kanakugiol' was recently isolated from Lindera erythrocarpa and showed free radical-scavenging and antifungal activities. However, the details of the anti-cancer effect of kanakugiol on breast cancer cells remain unclear. We investigated the effect of kanakugiol on the growth of MCF-7 human breast cancer cells. Kanakugiol affected cell cycle progression, and decreased cell viability in MCF-7 cells in a dose-dependent manner. It also enhanced PARP cleavage (50 kDa), whereas DNA laddering was not induced. FACS analysis with annexin V-FITC/PI staining showed necrosis induction in kanakugiol-treated cells. Caspase-9 cleavage was also induced. Expression of death receptors was not altered. However, Bcl-2 expression was suppressed, and mitochondrial membrane potential collapsed, indicating limited apoptosis induction by kanakugiol. Immunofluorescence analysis using α-tubulin staining revealed mitotic exit without cytokinesis (4N cells with two nuclei) due to kanakugiol treatment, suggesting that mitotic catastrophe may have been induced via microtubule destabilization. Furthermore, cell cycle analysis results also indicated mitotic catastrophe after cell cycle arrest in MCF-7 cells due to kanakugiol treatment. These findings suggest that kanakugiol inhibits cell proliferation and promotes cell death by inducing mitotic catastrophe after cell cycle arrest. Thus, kanakugiol shows potential for use as a drug in the treatment of human breast cancer.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.12
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• pp.7621-7628
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• 2013

Human epidermal growth factor receptor (HER) status is an important prognostic factor in breast cancer. There is no globally accepted method for determining its status, and which method is most precise is still a matter of debate. We here analyzed HER2 mRNA expression by quantitative reverse transcription-PCR (qRT-PCR) and HER2 DNA amplification using multiplex ligation-dependent probe amplification (MLPA). In parallel, we performed a routine evaluation of HER2 protein by immunohistochemistry (IHC). To assess the accuracy of the RT-PCR and MLPA techniques, a combination of IHC and fluorescence in situ hybridization (FISH) was used, substituting FISH when the results of IHC were ambiguous (2+) and for those IHC results that disagreed with MLPA and qRT-PCR, this approach being termed IHC-FISH. The IHC results for four samples were not compatible with the MLPA and qRT-PCR results; the MLPA and qRT-PCR results for these samples were confirmed by FISH. The correlations between IHC-FISH and qRT-PCR or MLPA were 0.945 and 0.973, respectively. The ASCO/CAP guideline IHC/FISH correlation with MLPA was (0.827) and with RT-PCR was (0.854). The correlations between the IHC results (0, 1+ as negative, and 3+ as positive) and qRT-PCR and MLPA techniques were 0.743 and 0.831, respectively. Given the shortcomings of IHC analysis and greater correlations between MLPA, qRT-PCR, and FISH methods than IHC analysis alone with each of these three methods, we propose that MLPA and real-time PCR are good alternatives to IHC. However a suitable cut-off point for qRTPCR is a prerequisite for determining the exact status of HER2.

• Journal of Microbiology and Biotechnology
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• v.10 no.1
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• pp.27-34
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• 2000

The chloroform and methanol (2;1, v/v) extract from an edible plant, Actinidia arguta Planchon, appeared to possess antitumor activity against human leukemias Jurkat T and U937 cells through inducing apoptosis. The substance in the solvent extract was purified by silica gel column chromatography, preparative TLC, and Sephadex LH-20 column chromatography. Characteristics of the substance analyzed by UV scanning analysis, $^1H$ and $^{13}C$ NMR spectra suggested that the substance belongs to the chlorophyll derivatives-like group. The $IC_{50}$ value of the chlorophyll derivative (Cp-D) determined by MTT assay was $15\mu\textrm{g}/ml$ for Jurkat, $10\mu\textrm{g}/ml$ for U937, and $11.4\mu\textrm{g}/ml$ for HL-60m and was more toxic to these leukemias than to solid tumors or normal fibroblast. In order to elucidate cellular mechanisms underlying the cytotoxicity, the effect of the Cp-D on Jurkat T cells was investigated. When cells were treated with the Cp-D at a concentration of $15\mu\textrm{g}/ml$, [3H]thymidine incorporation declined rapidly and wa undetectable in 1h. However, no significant changes were made in the cell cycle distribution of the cells by 24h. The sub-Gl peak representing apoptotic cells began to be detectable in 36h, at which time apoptotic DNA fragmentation was also detected on agarose gel electrophoresis, demonstrating that the cytotoxic effect of the Cp-D is attributable to the induced apoptosis. Under the same conditions, although the protein level of cyclin-dependent kinases such as cdc4, csk6, cdk2, and cdc2 was not significantly changed until 24h, the kinase activity of all c안 rapidly declined and reached a minimum level within 1-6h and then recovered to the initial level by 12h and sustained until 24h. These results suggest that inactivation of cdks at an inappropriate time during the cell cycle progression in jurkat T cells following a treatment with the Cp-D leads to induction of apoptotic cell death.

• The Korean Journal of Physiology and Pharmacology
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• v.9 no.6
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• pp.333-339
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• 2005

B/K protein is a novel protein containing double C2-like domains. We examined the specific signaling pathway that regulates the transcription of B/K in PC12 cells. When the cells were treated with forskolin ($50{\mu}M$), B/K mRNA and protein levels were time-dependently decreased, reaching the lowest level at 3 or 4 hr, and thereafter returning to the control level. Chemicals such as dibutyryl-cAMP, cellpermeable cyclic AMP (cAMP) analogue and CGS21680, adenosine receptor $A_{2A}$ agonist, also repressed the B/K transcription. However, 1,9-dideoxyforskolin did not show inhibitory effect on B/K transcription, suggesting direct involvement of cAMP in the forskolin-induced inhibition of B/K transcription. Effect of forskolin, dibutyryl cAMP and CGS21680 was significantly reduced in PKA-deficient PC12 cell line (PC12-123.7). One cAMP-response element (CRE)-like sequence (B/K CLS) was found in the promoter region of B/K DNA, and electrophoretic mobility shift assay indicated its binding to CREM and CREB. Forskolin significantly suppressed the promoter activity in CHO-K1 cells transfected with the constructs containing B/K CLS, but not with the construct in which B/K CLS was mutated (AC：TG). Taken together, we suggest that the transcription of B/K gene in PC12 cells may be regulated by PKA-dependent mechanism.

• Korean Journal of Soil Science and Fertilizer
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• v.45 no.5
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• pp.829-835
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• 2012

The cultivation of genetically modified (GM) crops has increased due to their economic and agronomic advantages. Before commercialization of GM crops, however, we must assess the potential risks of GM crops on human health and environment. The aim of this study was to investigate the possible impact of Bt rice on the soil microbial community. Microbial communities were isolated from the rhizosphere soil cultivated with Bt rice and Nakdong, parental cultivar and were subjected to be analyzed using both culture-dependent and molecular methods. The total counts of bacteria, fungi, and actinomycetes in the rhizosphere of transgenic and conventional rice were not significantly different. Denaturing gradient gel electrophoresis (DGGE) analysis of PCR-amplified 16S rRNA genes revealed that the bacterial community structures during cultural periods were very similar each other. Analysis of dominant isolates in the rhizosphere cultivated with Bt and Nakdong rice showed that the dominant isolates from the soil of Bt rice and Nakdong belonged to the Proteobacteria, Cloroflexi, Actinobacteria, Firmicutes, and Acidobacteria. These results indicate that the Bt rice has no significant impact on the soil microbial communities during cultivation period. Further study remains to be investigated whether the residue of Bt rice effect on the soil environment.

• Asian Pacific Journal of Cancer Prevention
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• v.17 no.8
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• pp.3971-3977
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• 2016

Background: Breast cancer, the commonest cancer among women in the world, ranks top in India with an incidence rate of 1,45,000 new cases and mortality rate of 70,000 women every year. Chemotherapy outcome for breast cancer is hampered due to poor response and irreversible dose-dependent cardiotoxicity which is determined by genetic variations in drug metabolizing enzymes and transporters. Pregnane X receptor (PXR), a member of the nuclear receptor superfamily, induces expression of drug metabolizing enzymes (DMEs) and transporters leading to regulation of xenobiotic metabolism. Materials and Methods: A genomic region spanning PXR 3' UTR was amplified and sequenced using genomic DNA isolated from 96 South Indian breast cancer patients. Genetic variants observed in our study subjects were queried in miRSNP to establish SNPs that alter miRNA binding sites in PXR 3' UTR. In addition, enrichment analysis was carried out to understand the network of miRNAs and PXR in drug metabolism using DIANA miRpath and miRwalk pathway prediction tools. Results: In this study, we identified SNPs rs3732359, rs3732360, rs1054190, rs1054191 and rs6438550 in the PXR 3; UTR region. The SNPs rs3732360, rs1054190 and rs1054191 were located in the binding site of miR-500a-3p, miR-532-3p and miR-374a-3p resulting in the altered PXR level due to the deregulation of post-transcriptional control and this leads to poor treatment response and toxicity. Conclusions: Genetic variants identified in PXR 3' UTR and their effects on PXR levels through post-transcriptional regulation provide a genetic basis for interindividual variability in treatment response and toxicity associated with chemotherapy.