• Tuberculosis and Respiratory Diseases
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• v.65 no.6
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• pp.476-486
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• 2008

Background: TRAIL (TNF-related apoptosis inducing ligand) is a newly identified member of the TNF gene family which appears to have tumor-selective cytotoxicity due to the distinct decoy receptor system. TRAIL has direct access to caspase machinery and induces apoptosis regardless of p53 phenotype. Therefore, TRAIL has a therapeutic potential in lung cancer which frequently harbors p53 mutation in more than 50% of cases. However, it was shown that TRAIL also could activates $NF-{\kappa}B$ in some cell lines which might inhibit TRAIL-induced apoptosis. This study was designed to investigate whether TRAIL can activate $NF-{\kappa}B$ in lung cancer cell lines relatively resistant to TRAIL-induced apoptosis and inhibition of $NF-{\kappa}B$ activation using proteasome inhibitor MG132 which blocks $I{\kappa}B{\alpha}$ degradation can sensitize lung cancer cells to TRAIL-induced apoptosis. Methods: A549 (wt p53) and NCI-H1299 (null p53) lung cancer cells were used and cell viability test was done by MTT assay. Apoptosis was confirmed with Annexin V assay followed by FACS analysis. To study $NF-{\kappa}B$-dependent transcriptional activation, a luciferase reporter gene assay was used after making A549 and NCI-H1299 cells stably transfected with IgG ${\kappa}-NF-{\kappa}B$ luciferase construct. To investigate DNA binding of $NF-{\kappa}B$ activated by TRAIL, electromobility shift assay was used and supershift assay was done using anti-p65 antibody. Western blot was done for the study of $I{\kappa}B{\alpha}$ degradation. Results: A549 and NCI-H1299 cells were relatively resistant to TRAIL-induced apoptosis showing only 20~30% cell death even at the concentration 100 ng/ml, but MG132 ($3{\mu}M$) pre-treatment 1 hour prior to TRAIL addition greatly increased cell death more than 80%. Luciferase assay showed TRAIL-induced $NF-{\kappa}B$ transcriptional activity in both cell lines. Electromobility shift assay demonstrated DNA binding complex of $NF-{\kappa}B$ activated by TRAIL and supershift with p65 antibody. $I{\kappa}B{\alpha}$ degradation was proven by western blot. MG132 completely blocked both TRAIL-induced $NF-{\kappa}B$ dependent luciferase activity and DNA binding of $NF-{\kappa}B$. Conclusion: This results suggest that inhibition of $NF-{\kappa}B$ can be a potentially useful strategy to enhance TRAIL-induced tumor cell killing in lung cancer.

• BMB Reports
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• v.30 no.3
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• pp.205-210
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• 1997

p53 tumor suppressor plays an important role in the regulation of cellular proliferation. To identify proteins regulating the expression of p53 in rat liver, we analyzed p53 promoter by electrophoretic mobility shift assay (EMSA) and DNase I footprinting assay. We found that a protein binds the sequence CACGTG, bHLH consensus sequence in rat p53 promoter. Southwestern blotting analysis with oligonucleotides containing this sequence shows that the molecular weight of the protein is 100 kDa. This size is not compatible with the bHLH family such as USF or c-Myc/Max which is known to regulate the expression of the human and mouse p53 gene. Therefore this 100 kDa protein may be a new protein regulating basal transcription of rat p53. We purified this 100 kDa protein through sequence-specific DNA affinity chromatogaphy.

• Journal of Korean Neurosurgical Society
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• v.37 no.1
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• pp.48-53
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• 2005

Objective: The purpose of this study is to elucidate in vitro responses to combined gamma knife irradiation and p53 gene transfection on human malignant glioma cell lines. Methods: Two malignant human glioma cell lines, U87MG (p53-wild type) and U373MG (p53-mutant) were transfected with an adenoviral vector containing p53 (MOI of 50) before and after applying 20Gy of gamma irradiation. Various assessments were performed, including, cell viability by MTT assay; apoptosis by annexin assay; and cell cycle by flow cytometry, for the seven groups: mock, p53 only, gamma knife (GK) only, GK after LacZ, LacZ after GK, GK after p53, p53 after GK. Results: Cell survival decreased especially, in the subgroup transfected with p53 after gamma irradiation. Apoptosis tended to increase in p53 transfected U373 MG after gamma irradiation (apoptotic rate, 38.9%). The G2-M phase cell cycle arrest markedly increased by transfecting with p53, 48 hours after gamma knife irradiation in U373 MG (G2-M phase, 90.8%). Conclusion: These results suggest that the in vitro effects of combined gamma knife irradiation and p53 gene transfection is an augmentation of apoptosis and G2-M phase cell cycle arrest, which are more exaggerated in U373 MG with p53 transfection after gamma knife irradiation.

• Tuberculosis and Respiratory Diseases
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• v.56 no.2
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• pp.187-197
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• 2004

Background : Nitric Oxide (NO) is a multi-faceted molecule with dichotomous regulatory roles in many areas of biology. NO can promote apoptosis in some cells, whereas it inhibits apoptosis in other cell types. This study was performed to characterize NO-induced cell death in lung epithelial cells and to investigate the roles of cell death regulators including iron, bcl-2 and p53. Methods : A549 cells were used for lung epithelial cells. SNP (sodium nitroprusside) and SNAP (S-nitroso-N-acetyl- penicillamine) were used for NO donor. Cytoxicity assay was done by MTT assay and crystal violet assay. Apoptotic assay was done by fluorescent microscopy after double staining with propidium iodide and hoecst 33342. Iron inhibition study was done with RBCs and FeSO4. For bcl-2 study, bcl-2 overexpressing cells (A549-bcl-2) were used and for p53 study, Western blot analysis and p53 functionally knock-out cells (A549-E6) were used. Results : SNP and SNAP induced dose-dependent cell death in A549 cells and fluorescent microscopy revealed that SNAP induced apoptosis in low doses but necrosis in high doses while SNP induced exclusively necrotic cell death. Iron inhibition study using RBCs and FeSO4 significantly blocked SNAP-induced cell death. And also SNAP-induced cell death was blocked by bcl-2 overexpression. Finally, we found that SNAP activate p53 by Western blot analysis and that SNAP-induced cell death was decreased in the abscence of p53. Conclusion : In lung epithelial cells, NO can induce cell death, more precisely apoptosis in low doses and necrosis in high doses. And iron, bcl-2, and p53 play important roles in NO-induced cell death.

• Journal of the Korean Society of Food Science and Nutrition
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• v.46 no.4
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• pp.417-425
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• 2017

Cnidium monnieri (L.) Cusson is distributed in China and Korea, and the fruit of C. monnieri is used as traditional Chinese medicine to treat carbuncle and pain in female genitalia. In this study, we examined the anti-proliferation and cell cycle arrest effects of ethanol extracts from C. monnieri (CME) in AGS gastric cancer cells. Our results show that CME suppressed cell proliferation and induced release of lactate dehydrogenase (LDH) in AGS cells by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay and LDH assay. Cell morphology was altered by CME in a dose-dependent manner. In order to identify the cell cycle arrest effects of CME, we investigated cell cycle analysis after CME treatment. In our results, CME induced cell cycle arrest at G1 phase. Protein kinase B (Akt) plays a major role in cell survival mechanisms such as growth, division, and metastasis. Akt protein regulates various downstream proteins such as glycogen synthase kinase-$3{\beta}$ (GSK-$3{\beta}$) and tumor protein p53 (p53). Expression levels of p-Akt, p-GSK-$3{\beta}$, p53, p21, cyclin E, and cyclin-dependent kinase 2 (CDK2) were determined by Western blot analysis. Protein levels of p-Akt, p-GSK-$3{\beta}$, and cyclin E were reduced while those of p53, p21, and p-CDK2 (T14/Y15) were elevated by CME. Moreover, treatment with CME, LY294002 (phosphoinositide 3-kinase/Akt inhibitor), BIO (GSK-$3{\beta}$ inhibitor), and Pifithrin-${\alpha}$ (p53 inhibitor) showed that cell cycle arrest effects were mediated through regulation of the Akt/GSK-$3{\beta}$/p53 signaling pathway. These results suggest that CME induces cell cycle arrest at G1 phase via the Akt/GSK-$3{\beta}$/p53 signaling pathway in AGS gastric cancer cells.

• BMB Reports
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• v.32 no.1
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• pp.45-50
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• 1999

We previously found three transcription factor-binding motifs in the rat p53 promoter. They are two recognition motifs of NF1-like protein (NF1-like element 1: -296 ~ -312, NF1-like element 2: -195 ~ -219) and a bHLH protein binding element (-142 ~ -146). In this study, we investigated the DNA-protein complex formation of the three elements with nuclear extracts from both normal and regenerating liver to find the element involved in the induced transcription of p53. The level of each DNA-protein complex on NF1-like and bHLH motifs was not changed. Instead, a new element located at -264 ~ -284 was detected in the DNase I footprinting assay with regenerating nuclear extract. This element has partial homology to the AP1 consensus motif. However, the competition studies with diverse oligonucleotides suggest that the binding protein is not AP1. An in vitro transcription assay shows that this element is important for the transcriptional activation of the rat p53 promoter. Therefore, for the induced transcription of the rat p53 promoter, the-264 ~ -284 region is required in addition to two NF1-like and one bHLH motif.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.8
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• pp.3731-3736
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• 2014

Phytic acid (PA) has been reported to have positive nutritional benefits and prevent cancer formation. This study investigated the anticancer activity of rice bran PA against hepatocellular carcinoma (HepG2) cells. Cytotoxicty of PA (0.5 to 4mM) was examined by MTT and LDH assays after 24 and 48h treatment. Apoptotic activity was evaluated by expression analysis of apoptosis-regulatory genes [i.e. p53, Bcl-2, Bax, Caspase-3 and -9] by reverse transcriptase-PCR and DNA fragmentation assay. The results showed antioxidant activity of PA in Fe3+ reducing power assay ($p{\leq}0.03$). PA inhibited the growth of HepG2 cells in a concentration dependent manner ($p{\leq}0.04$). After 48h treatment, cell viability was recorded 84.7, 74.4, 65.6, 49.6, 36.0 and 23.8% in MTT assay and 92.6, 77.0%, 66.8%, 51.2, 40.3 and 32.3% in LDH assay at concentrations of 1, 1.5, 2.0, 2.5, 3.0, and 3.5mM, respectively. Hence, treatment of PA for 24h, recorded viability of cells 93.5, 88.6, 55.5, 34.6 and 24.4% in MTT assay and 94.2, 86.1%, 59.7%, 42.3 and 31.6%, in LDH assay at concentrations of 1, 2.2, 3.0, 3.6 and 4.0mM, respectively. PA treated HepG2 cells showed up-regulation of p53, Bax, Caspase-3 and -9, and down-regulation of Bcl-2 gene ($p{\leq}0.01$). At the $IC_{50}$ (2.49mM) of PA, the p53, Bax, Caspase-3 and-9 genes were up-regulated by 6.03, 7.37, 19.7 and 14.5 fold respectively. Also, the fragmented genomic DNA in PA treated cells provided evidence of apoptosis. Our study confirmed the biological activity of PA and demonstrated growth inhibition and induction of apoptosis in HepG2 cells with modulation of the expression of apoptosis-regulatory genes.

• Tuberculosis and Respiratory Diseases
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• v.45 no.2
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• pp.333-340
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• 1998

Background: Lung cancer is the leading cause of cancer over the world. P53 alteration is by far the most common genetic defect in lung cancer. The mutation of p53 protein involves the loss of inhibitory function of p53 related tumor suppressor gene and resultant oncogenesis. The analysis of p53 alterations consists of immunohistochemical stain, PCR based assay, or serologic ELISA (enzyme-linked immunosorbent assay). Methods : Serum levels of p53 mutant protein were measured in 69 cases of lung cancer (adenocarcinoma n=29, epidermoid n=16, small cell n=13, large cell n=1, undifferentiated n=1, undetermined n=9) and 42 controls of respiratory disorders using ELISA. Immunohistochemical stain in tissue was performed using monoclonal antibody of p53 in lung cancer subjects. Results: Both serum p53s in nonsmall cell cancer ($0.28{\pm}0.44ng/ml$) and in small cell cancer ($0.20{\pm}0.14ng/ml$) were not different from controls ($0.34{\pm}0.20ng/ml$). Also there was no significant difference in serum p53 according to tumor stages. P53 immunohistochemical stain showed 50% positivity overall in lung cancer. There were no close correlation between serologic level and positivity of immunohistochemical stain. Conclusion: The serologic determination of p53 mutant protein is thought to have no diagnostic role in lung cancer. Immunohistochemical stain in lung cancer specimen shows 50% positivity.

• IMMUNE NETWORK
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• v.1 no.2
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• pp.151-161
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• 2001

Background: Inactivation in p53 tumor suppressor gene through a point mutation and deletion is one of the most frequent genetic changes found in human cancer, with 50% of an incidence. This high rate of mutation mostly suggests that the gene plays a central role in the development of cancer and the mutations detected so far were found in exons 5 to 8. Mutation of p53 locus produced accumulation of abnormal p53 protein, and negative regulation of cell proliferation and transcriptional activation as a suppressor of transformation were lost. In addition, inhibition of its normal cellular function of wild-type by mutant is an important step in tumorigenesis. Method: 4 colon cancer cell lines (SNU C1, C2A, C4, C5) were examined for mutation in exons 5 to 8 of the p53 tumor suppressor gene by PCR-SSCP analysis and expression pattern by western blotting and immunoprecipitation. p53-mediated transactivation ability were examined by CAT assay and base substitution of p53 in SNU C2A cell were detected by DNA sequencing. Results: 1) SNU C2A cell and SNU C5 cell were detected mobility shifts each in exon 5 and exon 7 of p53 gene by the PCR-SSCP method, implicating being of p53 mutation. 2) 3 colon cancer cell lines (SNU C1, SNU C2A, SNU C5) expressed wild type and mutant type p53 protein. 3) In northern blot experiment, SNU C2A and SNU C5 cell expressed high level of p53 mRNA. 4) Results of p53-mediated transactivation in colon cancer cell lines by CAT assay represented only SNU C2A cell has transcriptional activity. 5) DNA sequencing in SNU C2A cell showed missense mutation in codon 179 of one allele, histidine to arginine and wild type p53 in the other allele. Conclusion: Colon cancer cell lines showed correlation with mutation in p53 gene and accumulation of abnormal p53 protein. Colon cancer cell SNU C2A retained p53-mediated transactivation as heterozygous p53 with one mutant allele in 179 codon and the other wild-type allele.

• Tuberculosis and Respiratory Diseases
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• v.53 no.3
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• pp.275-284
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• 2002

Background : Gemcitabine is a new anti-cancer agent for treating non-small cell lung cancer. Functioning as an antimetabolite, it induces anti-cancer effects by suppressing DNA synthesis after being incorporated into the DNA as a cytosine arabinoside analogue. When Gemcitabine is incorporated into the DNA, the p53 gene may be activated by induction of the DNA defect. However, there are a few studies on the molecular mechanisms of Gemcitabine-induced cell death. This study examined the role of p53 in Gemcitabine-induced cell death. Methods : A549 and NCl-H358 lung cancer cells were used in this study. The cell viability test was done using a MTT assay at Gemcitabine concentrations of 10nM, 100nM, 1uM, 10uM and 100uM. A FACScan analysis with propium iodide staining was used for the cell cycle analysis. Western blot analysis was done to investigate the extent of p53 activation. For the functional knock-out of p53, stable A549-E6 cells and H358-E6 cells were transfected pLXSN-16E6SD which is over expresses the human papilloma virus E6 protein that constantly degrades p53 protein. The functional knock out of p53 was confirmed by Western blot analysis after treatment with a DNA damaging agent, doxorubicine. Results : Gemcitabine exhibited cell toxicity in dose-dependent fashion. The cell cycle analysis resulted in an S phase arrest. Western blot analysis significant p53 activation in time-dependent manner. Gemcitabine-induced cytotoxicity was reduced by 20-30% in the A549-E6 cells and the 30-40% in H358-E6 cells when compared with the A549-neo and H358-neo control cells. Conclusion : Gemcitabine induces an S phase arrest, as expected for the anti-metabolite, and activates the p53 gene, Furthermore, p53 might play an important role in Gemcitabine-induced cell death. Further investigation into the molecular mechanisms on how Gemcitabine activates the p53 gene and its signaling pathway are recommended.