• The Korean Journal of Physiology and Pharmacology
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• v.18 no.1
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• pp.73-78
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• 2014

Cell death and survival are tightly controlled through the highly coordinated activation/inhibition of diverse signal transduction pathways to insure normal development and physiology. Imbalance between cell death and survival often leads to autoimmune diseases and cancer. Death receptors sense extracellular signals to induce caspase-mediated apoptosis. Acting upstream of CED-3 family proteases, such as caspase-3, Bcl-2 prevents apoptosis. Using short hairpin RNAs (shRNAs), we suppressed Bcl-2 expression in Jurkat T cells, and this increased TCR-triggered AICD and enhanced TNFR gene expression. Also, knockdown of Bcl-2 in Jurkat T cells suppressed the gene expression of FLIP, TNF receptor-associated factors 3 (TRAF3) and TRAF4. Furthermore, suppressed Bcl-2 expression increased caspase-3 and diminished nuclear factor kappa B (NF-${\kappa}B$) translocation.

• BMB Reports
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• v.54 no.3
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• pp.176-181
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• 2021

Bcl-x, a member of the Bcl-2 family, plays a key role in apoptosis. Alternative splicing of Bcl-x pre-mRNA through alternative 5' splice-site selection produces an anti-apoptotic mRNA isoform that includes exon 2b and a pro-apoptotic Bcl-x mRNA isoform that excludes exon 2b. Here we used Bcl-x minigene and identified SRSF2 and SRSF6 as two regulatory factors of 5' splice-site selection of Bcl-x pre-mRNA. We selected binding clusters closer to 5' splice-sites from multiple potential binding sites of SRSF2 and SRSF6 to perform loss of functions analysis through site-directed mutagenesis. Our results demonstrated that these mutations did not abolish regulatory functions of SRSF2 or SRSF6, indicating that a single binding motif or a cluster was not a functional target of these proteins in Bcl-x pre-mRNA splicing. Random deletion mutagenesis did not disrupt the role of SRSF2 and SRSF6. Importantly, mutagenesis of 5' splice-site to a conserved or a weaker score demonstrated that the weaker strength of the target 5' splice-site or higher strength of the other 5' splice-site strength limited the role of SRSF2 and SRSF6 in 5' splice-site activation.

• Journal of Photoscience
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• v.9 no.2
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• pp.225-228
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• 2002

Bcl-2 is a member of large bcl-2 family and protects cells from apoptosis. Using bcl-2-expressing adenovirus vector (Ad-bc1-2), we investigated the effect of bc1-2 on UVB-induced apoptosis. Adenovirus vector efficiently introduced bc1-2 gene in cultured normal mouse keratinocytes (NMK cells); almost all NMK cells (lx10$^{6}$ ) were transfected at Ixl0$^{8}$ PFU/ml. Bcl-2-transfected NMK cells were significantly resistant to UVB-induced apoptosis with the suppressive effect dependent on bcl-2-expression level. Following UVB irradiation caspase 8, 3, 9 activities were stimulated in NMK cells, while in bc1-2-transfected cells, only caspase 8, but not caspase 3 or 9 activities were stimulated. In order to investigate the effect of bc1-2 in vivo, topical application of Ad-bc1-2 on tape-stripped mouse skin was performed. Following the application, Bc1-2 was efficiently overexpressed in almost all viable keratinocytes. The expression was transient with the maximal expression of Bc1-2 at 1st day following the application of lxl0$^{9}$ PFU in 200ml. The introduced Bc1-2 remained at least for 6 days. UVB irradiation (1250 J/m$^2$) induced apoptosis within 12 h and the maximal effect was observed at 24 h in control mouse skin. Bc1-2-transfected mice skin were resistant to UVB-induced apoptosis. Topical application of empty adenovirus vector alone had no effect on Bc1-2 expression or UVB-induced apoptosis. These results indicate that adenovirus vector is an efficient gene delivery system into keratinocytes and that Bcl-2 is a potent inhibitor of UVB-induced apoptosis both in vitro and in vivo.

• Development and Reproduction
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• v.15 no.2
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• pp.159-166
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• 2011

Simazine (6-chloro-N,N'-diethyl-1,3,5-triazine-2,4-diamine) is a triazine herbicide that has been applied worldwide including Korea for agricultural purposes. Simazine is the second most commonly detected pesticide in surfaceand ground-water in the United States, Europe and Australia. It has been shown that simazine is a potent endocrine disruptor in wildlife and laboratory animals. Although many endocrine disruptors can induce apoptosis in various types of cells, the effects of simazine on apoptosis and on the expression of Bcl-2 family genes are not known. Also it is unknown the effect of simazine on the expression of steroidogenesis-regulating genes in testicular cells. In this study, we investigated the effect of simazine on the expression levels of apoptosis- and steroidogenesis-regulating genes in testicular cells. We found that a low concentration of simazine can alter the mRNA expression levels of steroidogenesis-related genes and Bcl-2 family genes in mouse Sertoli cells and rat Leydig cells. Thus, our results suggest that simazine can disturb normal testicular development and reproductive function by altering the expression of genes that are critical for the regulation of apoptosis and steroidogenesis.

• Clinical and Experimental Pediatrics
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• v.52 no.6
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• pp.696-700
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• 2009

Purpose : We screened more than 350 compounds with an endoperoxide ring structure in search of an anti-leukemic drug and found that compound 127 (c-127) could induce significant cytotoxicity in HL-60 cells. In this study, we investigated the molecular mechanisms of compound 127-induced antitumor activity on HL-60 cells. Methods : HL-60 cells were cultured in Rosewell Park Memorial Institute 1640 and cell viability was measured by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], a tetrazole assay. Apoptosis was assessed by a DNA fragmentation test. Apoptotic machineries were determined by Western blot analysis. Results : C-127 could induce a cytotoxic effect at 24 h and apoptosis at 6 h, which was demonstrated with MTT assay and DNA fragmentation test, respectively. The apoptotic effect of this drug was caused by the activation of the intracellular caspase-8,3 activation, the cleavage of pro-apoptotic Bid, and the increase of c-Jun expression accompanied with JNK (Jun N-terminal kinases) phosphorylation. On the contrary, it increased the expression of anti-apoptotic Bcl-2 levels, leading to the induction of the induction of anti-apoptotic effect. Taken together, the present study demonstrated that c-127 was a potent inducer of cytotoxicity on HL-60 cells through apoptotic mechanisms, which included the activation of caspase family, the regulation of Bcl-2 family, and the activation of JNK signaling pathway. Conclusion : Our results suggest that c-127 has a strong antitumor activity through the regulation of various apoptotic machineries on HL-60 cells. The compound may be utilized as an effective and potentially therapeutic drug in leukemia.

• Journal of Physiology & Pathology in Korean Medicine
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• v.22 no.3
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• pp.630-641
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• 2008

Gamisamgibopae-tang (GMSGBPT) is a traditional Korean medicine, which has been used for patients suffering from a lung disease in Oriental medicine. In the present study, we examined the biochemical mechanisms of apoptosis by GMSGBPT in NCI-H460 and A549 human non-small-cell lung cancer cell lines. It was found that GMSGBPT could inhibit the cell proliferation of A549 cells in a concentration-dependent manner, however GMSGBPT did not affect the cell proliferation of NCI-H460 cells. Apoptotic cell death in A549 cells were detected using DAPI staining and annexin V fluorescein methods. The induction of apoptotic cell death by GMSGBPT was connected with a down-regulation of anti-apoptotic Bcl-2 and Bcl-xL expression, and proteolytic activation of caspase-3 and caspase-9 in A549 cells. However, GMSGBPT did not affect the levels of pro-apoptotic Bax and Bad expression, and activity of caspase-8. GMSGBPT treatment also concomitant degradation and/or inhibition of poly (ADP-ribose) polymerase (PARP), ${\beta}$-catenin, phospholipase C-1 (PLC${\gamma}$1) and DNA fragmentation factor 45/inhibitor of caspase-activated DNase (DFF45/ICAD). Taken together, these findings suggest that GMSGBPT may be a potential chemotherapeutic agent for the control of human non-small-cell lung cancer cells and further studies will be needed to identify the active compounds that confer the anti-cancer activity of GMSGBPT.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.14
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• pp.6067-6071
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• 2015

Background: Gastric cancer accounts for about 8% of the total cancer cases and 10% of total cancer deaths worldwide. It is the second lethal cancer after esophageal cancer and is considered the fourth most common cancer in north and northwest Iran. The bcl2 family has a key role in the regulation of apoptosis and change in its expression can contribute to cancer. This study initially scheduled to determine the expression of bcl2 gene in tissue samples of adenocarcinoma cancer patients. Materials and Methods: A total of 10 samples of gastric adenocarcinoma and 10 of normal tissues from Sari hospital were selected and after DNA extraction from tissues, bcl2 gene expression assayed by real-time PCR. Results: Our results demonstrated higher expression of the bcl2 gene in control compared with cancer and marginal cancer tissues. Conclusions: On one hand BCL2 plays an important role as an oncogene to inhibit apoptosis; on the other hand, it can initiate cell cycle arrest at G0 stage. Our observed association between its expression and patient survival is quite conflicting and may be tissue-specific. The data suggest expression both tumoural and non-tumoral(marginal) groups have lowered expression than controls (P>0.05). Due to the low number of samples we could not examine the relationship with clinicopathological features. However, bcl-2 expression may be important for prognostic outcome or a useful target for therapeutic intervention.

• Journal of the Korean Society of Food Science and Nutrition
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• v.44 no.3
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• pp.338-346
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• 2015

This study was performed to elucidate the anti-proliferative and apoptotic mechanism of flavonoids in HT-29 human colon cancer cells. We investigated the anti-proliferative activity of flavonoids in HT-29 human colon cancer cells via cell viability assay (MTT assay), caspase-3 activity, RT-PCR, and western blotting. We cultured HT-29 cells in the presence of various flavonoids (apigenin, rutin, naringenin, and myricetin) at a concentration of $100{\mu}M$. In the MTT assay, naringenin showed the strongest effect on cell viability in HT-29 colon cancer cells. Caspase-3 activity, a marker of apoptosis, significantly increased upon naringenin treatment. For RT-PCR, myricetin significantly increased Bax protein levels, naringenin increased p53 protein levels, and rutin reduced expression of the anti-apoptotic protein Bcl-2. Western blotting of HT-29 colon cancer cells showed that myricetin increased cleaved caspase-3 protein levels, naringenin significantly increased poly (ADP-ribose) polymerase protein levels, and rutin increased E-cadherin protein levels. These results indicate that flavonoid exerts anticancer effects on human colon HT-29 cells through a caspase-dependent apoptotic pathway.

• International Journal of Oral Biology
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• v.34 no.1
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• pp.7-14
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• 2009

Nitric oxide (NO) acts as an intracellular messenger at the physiological level but can be cytotoxic at high concentrations. The cells within periodontal tissues, such as gingival and periodontal fibroblasts, contain nitric oxide syntheses and produce high concentrations of NO when exposed to bacterial lipopolysaccharides and cytokines. However, the cellular mechanisms underlying NO-induced cytotoxicity in periodontal tissues are unclear at present. In our current study, we examined the NO-induced cytotoxic mechanisms in human gingival fibroblasts (HGF). Cell viability and the levels of reactive oxygen species (ROS) were determined using a MTT assay and a fluorescent spectrometer, respectively. The morphological changes in the cells were examined by Diff-Quick staining. Expression of the Bcl-2 family and Fas was determined by RT-PCR or western blotting. The activity of caspase-3, -8 and -9 was assessed using a spectrophotometer. Sodium nitroprusside (SNP), a NO donor, decreased the cell viability of the HGF cells in a dose- and time-dependent manner. SNP enhanced the production of ROS, which was ameliorated by NAC, a free radical scavenger. ODQ, a soluble guanylate cyclase inhibitor, did not block the SNP-induced decrease in cell viability. SNP also caused apoptotic morphological changes, including cell shrinkage, chromatin condensation, and DNA fragmentation. The expression of Bax, a member of the proapoptotic Bcl-2 family, was upregulated in the SNP-treated HGF cells, whereas the expression of Bcl-2, a member of the anti-apoptotic Bcl-2 family, was downregulated. SNP augmented the release of cytochrome c from the mitochondria into the cytosol and enhanced the activity of caspase-8, -9, and -3. SNP also upregulated Fas, a component of the death receptor assembly. These results suggest that NO induces apoptosis in human gingival fibroblast via ROS and the Bcl-2 family through both mitochondrial- and death receptor-mediated pathways. Our data also indicate that the cyclic GMP pathway is not involved in NO-induced apoptosis.

• BMB Reports
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• v.41 no.1
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• pp.11-22
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• 2008

Apoptosis (programmed cell death) is a cellular self-destruction mechanism that is essential for a variety of biological events, such as developmental sculpturing, tissue homeostasis, and the removal of unwanted cells. Mitochondria play a crucial role in regulating cell death. $Ca^{2+}$ has long been recognized as a participant in apoptotic pathways. Mitochondria are known to modulate and synchronize $Ca^{2+}$ signaling. Massive accumulation of $Ca^{2+}$ in the mitochondria leads to apoptosis. The $Ca^{2+}$ dynamics of ER and mitochondria appear to be modulated by the Bcl-2 family proteins, key factors involved in apoptosis. The number and morphology of mitochondria are precisely controlled through mitochondrial fusion and fission process by numerous mitochondria-shaping proteins. Mitochondrial fission accompanies apoptotic cell death and appears to be important for progression of the apoptotic pathway. Here, we highlight and discuss the role of mitochondrial calcium handling and mitochondrial fusion and fission machinery in apoptosis.