• The Korean Journal of Physiology and Pharmacology
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• 제18권1호
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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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• 제54권3호
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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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• 제9권2호
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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.

• 한국발생생물학회지:발생과생식
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• 제15권2호
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• pp.159-166
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• 2011

Simazine은 triazine계 제초제로서, 잡초와 일년생 풀들을 통제하는데 우리나라를 포함하여 세계적으로 널리 사용되고 있으며, 미국, 유럽, 오스트레일리아의 경우, 물에서 두 번째로 많이 검출되는 살충제로 알려져 있다. Simazine에오염된 토양과 물을 통하여 사람에게 노출되어 이후 생체 내에 수년간 잔존하며 특히, 생물체의 지방 및 조직에 농축되는특징을 갖는 내분비계 장애물질로 분류되어졌다. 하지만 simazine이 정소세포의 사멸 또는 생존에 미치는 영향과 세포예정사를 관장하는 Bcl-2 family 유전자 발현에 미치는 영향을 보고한 연구는 없다. 또한 simazine이 성의 분화와 생식기관발달에 중요한 steroidogenesis 관련 유전자의 발현에 미치는 영향 분석에 관한 연구도 미흡한 실정이다. 따라서 본 연구진은 이번 연구에서 simazine이 mouse Sertoli 세포와 rat Leydig 세포에서 세포사멸을 관장하는 Bcl-2 family 유전자군의발현과 steroidogenesis 조절관련 유전자군의 발현 변화에 미치는 영향을 조사하였고, simazine이상기 유전자군의 정상적인 발현을 저해함을 확인하였다. 그러므로 본 연구의 결과로 미루어 볼 때, 사람이나 기타 동물 등이 simazine에 장시간 노출될 경우, 생식 세포에 apoptosis가 유도될 수 있는 가능성을 제시하였으며, steroidogenesis 조절관련 유전자군의 발현을 교란시킴으로써, 정상적인 testis의 발달을 저해하여 남성 생식계의 기능장애를 유도할 가능성이 있음을 제시하였다.

• Clinical and Experimental Pediatrics
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• 제52권6호
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• pp.696-700
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• 2009

목 적 : Artemisinin은 인체에 대한 부작용이 적고 항말라리아 효능 뿐 아니라 강력한 항암효과가 있음이 알려져 있다. 저자들은 오까야마 약학대학에서 합성 된 350여개의 endoperoxide ring구조를 가진 artemisinin 유도체를 선별검사 하여 HL-60세포 주에 강력한 세포독성을 보여 준 c-127의 세포고사 유도 여부와 그 분자유전학적 기전을 알아보고자 하였다. 방 법 : HL-60세포는 RPMI 1640배지로 배양하였고 세포 생존율은 MTT분석으로 측정하였다. 세포고사 여부는 DNA추출과 전기영동법으로 확인하였으며 세포고사 유도 기전은 western blotting을 시행하여 알아보았다. 결 과 : C-127이 세포고사를 유도하여 HL-60세포의 세포 생존력을 농도 의존적으로 감소시켰다. C-127의 이런 항암효과의 분자 유전학적 기전으로는 caspase-8,3 활성화, Bcl-2 family인 Bid분절, JNK 인산화와 c-Jun 발현이 관여하였다. 결 론 : C-127이 HL-60 세포에서 세포고사를 유도하여 강력한 항암효과를 발현하였고, 그 기전으로 caspase cascade, Bcl-2 family, JNK인산화와 c-Jun활성화가 관여하였다.

• 동의생리병리학회지
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• 제22권3호
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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.

• 한국식품영양과학회지
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• 제44권3호
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• pp.338-346
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• 2015

본 연구에서는 최근 식생활의 서구화로 인해 발병률이 급증하고 있는 대장암의 진행을 억제하거나 감소시키고 인체 대장암 세포인 HT-29의 증식을 억제하며, 세포사멸을 유도하는 천연소재를 알아보기 위해서 flavonoid가 HT-29 인체 대장암 세포의 apoptosis 유도 및 기전에 미치는 영향을 알아보았다. MTT assay 결과 apigenin, rutin, naringenin, myricetin을 $100{\mu}M$ 농도로 처리하였을 때 62.71, 75.78, 74.24, 77.61%로 이 중 naringenin이 대장암 세포 성장에 억제 효과가 가장 높은 실험 결과를 나타내었다. Caspase-3 activity에서는 naringenin이 241.46%로 가장 높은 활성을 나타내었다. 이를 바탕으로 세포사멸과 관련된 유전자를 확인하고자 대장암 세포에 flavonoid인 apigenin, rutin, naringenin, myricetin에 $100{\mu}M$ 농도로 처리한 후 RTPCR을 실시한 결과, 세포사멸의 주요한 조절인자인 Bcl-2 family 단백질 중 Bcl-2는 rutin에 의해 감소되었고 Bax는 myricetin에 의해 증가하였으며, p53은 naringenin이 높게 발현되었다. 또한 western blotting을 통해 flavonoid인 apigenin, rutin, naringenin, myricetin에 $100{\mu}M$ 농도로 처리한 결과, Bcl-2 family 단백질과 더불어 세포사멸 조절에 중요한 역할을 하는 활성형인 cleaved caspase-3은 모두 증가하였고, 그중 myricetin이, PARP은 naringenin, E-cadherin은 rutin이 각각 높은 발현 양상을 나타내었다. 이번 실험 결과를 통해 flavonoid가 세포사멸의 주요한 조절 인자인 Bcl-2 family 단백질의 발현이나 caspase의 활성 등을 조절하여 암세포 사멸인자인 Bcl-2의 발현은 감소시키고 Bax, p53, PARP의 발현을 증가시키는 것을 통해 대장암 세포의 apoptosis를 유도하였다. 또한 암세포의 전이와 관련된 E-cadherin의 발현도 조절하는 것을 관찰하였다. 이상의 연구를 통해 flavonoid가 대장암 세포의 증식을 억제하는 효과가 있음을 확인하였으며, 세포사멸과 관련된 기전을 규명하였다. 이를 기초자료로 일상에서 쉽게 섭취할 수 있는 식품에 많이 존재하며 비교적 독성과 부작용이 적은 flavonoid를 이용한 천연 항암제 개발 가능성을 제시하였고, 추후 대장암의 암예방제 및 암치료제로 개발될 수 있도록 추가 연구 수행이 필요할 것으로 사료된다.

• Asian Pacific Journal of Cancer Prevention
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• 제16권14호
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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.

• International Journal of Oral Biology
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• 제34권1호
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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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• 제41권1호
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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.