• Journal of Gastric Cancer
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• v.4 no.4
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• pp.268-271
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• 2004

Purpose: Most gastrointestinal stromal tumors (GISTs) have gain-of-function mutations of the KIT or the platelet-derived growth factor receptor alpha (PDGFRA) genes, but approximately $10\%$ of the GISTs are wild types for both the KIT and the PDGFRA genes. The purpose of this study was to investigate the possibility that epidermal growth factor receptor (EGFR) gene mutation might be responsible for the pathogenesis of GIST. Materials and Methods: We analyzed the EGFR gene in 60 GISTs for the detection of somatic mutations by using the polymerase chain reaction (PCR), the single strand conformation polymorphism (SSCP), and DNA sequencing in exon 18, 19, and 21 encoding the kinase domain. Results: The SSCP analysis revealed no evidence of EGFR mutations in exon 18, 19, and 21 in GISTs. Conclusion: The data indicate that the EGFR gene may not be mutated in human GIST and suggest that therapies targeting the mutated EGFR gene products might not be useful in the treatment of GISTs.

• Journal of Periodontal and Implant Science
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• v.34 no.4
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• pp.807-818
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• 2004

산화질소는 생리적 농도에서 세포내 신호전달자로 작용하지만 높은 농도에서는 세포독성을 일으킨다. 최근 치은 섬유아세포와 치주인대 섬유아세포는 산화질소 합성효소를 가지고 있고 세균의 lipopolysaccharide나 cytokine에 의해 대량의 높은 농도의 산화질소가 합성된다는 보고가 있음에도 지금까지 치은 조직에서 산화질소의 세포독성에 대한 연구는 아직 이루어 지지않고 있다. 본 연구는 사람의 치은 섬유아세포에서, 산화질소유도세포 고사기전을 밝히는데 목적이 있다. 세포 생장력은 MTT 방법으로 측정하였고, 세포의 형태적 변화는 Diff-Quick 염색법으로 조사하였다. Bcl-2 famly와 Fas 발현 정도는 RT-PCR 방법에 의해 확인하였으며, caspase-3, -8 와 -9의 활성은 spectrophotometer로 reactive oxygen species (ROS)는 형광분광계에 의해 측정되었다. 미토콘드리아에서 세포질로 분비된 cytochrome c는 western blot으로 조사하였다. 산화질소 유리제인 sodium nitroprusside (SNP) 처리는 사람 섬유아세포의 생존률을 시간과 농도 의존적으로 감소시켰고, 세포용적축소, 염색사 용축, DNA 절편화를 일으켰다. 또한, SNP 처리로 미토콘드리아에서 세포질로 유리되는 cytochrome c 양이 증가되었고, caspase-9 과 caspase-3 의 활성이 증가되었다. 한편, SNP 처리에 의해 death receptor 구성요소인 Fas 발현이 증가되었고, caspase-8의 활성이 증가되었다. Bcl-2 family 에 대한 RT-PCR 분석결과, 세포고사를 억제하는 Bcl-2 발현은 감소되었으나 세포고사를 자극하는 Bax와 Bid의 발현은 증가되었다. Soluble guanylate cyclase 억제제인 ODQ는 SNP에 의한 세포 생존율 감소를 차단하지 못했다. 따라서, 본 실험의 결과들은 사람 섬유아세포에서 산화질소유도 세포고사에 Bcl-2 family나 ROS가 매개하는 미토콘드리아 의존 및 death receptor 의존 세포고사기전이 관여함을 시사하였다.

• Journal of Acupuncture Research
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• v.18 no.1
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• pp.129-145
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• 2001

Objectives : To characterize the antitumorigenic potential of three representative bee venom components, Melittin, Apamin, and Phospholipase A2, their effects on cell proliferation and apotosis of the human melanoma cell line SK-MEL-2 were analyzed using molecular biological approaches. Methodes & Results : To determine the doses of the drugs that do not induce cytotoxic damage to this cell line, cell viability was examined by MTT assay. While SK-MEL-2 cells treated with 0.5 - 2.0㎍/㎖ of each drug showed no recognizable cytotoxic effect, marked reductions of cell viability were detected at concentrations over 5.0㎍/㎖. [3H]thymidine incorporation assay for cell proliferation demonstrated that DNA replication of SK-MEL-2 cells is inhibited by Apamin and Phospholipase A2 in a dose-dependent manner. Consistent with this result, the cells were accumulated at the G1 phase of the cell cycle after treatment with Apamin and Phospholipase A2, whereas no detectable change in cell proliferation was identified by Melittin treatment. In addition, tryphan blue exclusion and flow cytometric analyses showed that all of these drugs can trigger apoptotic cell death of SK-MEL-2, suggesting that Melittin, Apamin, and Phospholipase A2 have antitumorigenic potential through the suppression of cell growth and/or induction of apoptosis. Qantitative RT-PCR analysis revealed that Apamin and Phospholipase A2 inhibit expression of growth-promoting genes such as c-Jun, c-Fos, and Cyciin D1. Furthermore, Phospholipase A2 induced tumor suppressors p53 and p21/Wafl. In addition, all three drugs were found to activate expression of a representative apoptosis-inducing gene Bax while expression of apoptosis-suppressing Bcl-2 and Bcl-XL genes was not changed. Taken together, this study strongly suggests that Metittin, Apamin, and Phosphalipase A2 may have antitumorigenic activities, which are associated with its growth-inhibiting and/or apoptosis-inducing potentials.

• International Journal of Oral Biology
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• v.32 no.2
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• pp.79-84
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• 2007

Oral squamous carcinoma (OSC) is the most common malignant neoplasm of the oral mucosa. Although the etiology of OSC is not fully understood, accumulated evidences indicate that the activation of proto-oncogenes and the inactivation of tumor suppressor genes underlie the disease development. An OSC cell line, YD-9 was newly established and characterized. However, the mutational analysis of p53 gene was not performed. Thus, in this study, the presence of mutation in the p53 gene was examined by amplification of exon-4 to -8 and subsequent DNA sequencing. Two point mutations were found in exon-4 and -6: A to G, resulting in amino acid change Tyr to Cys in exon-4, and C to G, resulting in amino acid change Gly to Arg in exon-6, respectively. Any mutation was not found in the exon-5, -7 and -8. The presented results would contribute to basic research to understand the biological mechanism of OSC using YD-9 cells.

• Journal of Gastric Cancer
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• v.3 no.3
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• pp.145-150
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• 2003

Purpose: The p53 tumor suppressor gene is believed to play a pivotal role in preventing the uncontrolled cellular growth characteristic of cancer. Mutation of the p53 gene represent one of the most common genetic alterations in human cancers, and the acquisition of such defects is strongly associated with tumor progression and metastasis. The aim of this study was to evaluate the relation between p53 immunoreactivity and the mutation of p53 gene in gastric adenocarcinoma obtained by laser capture microscope. Materials and Methods: Formalin fixed paraffin embedded tissue specimens were obtained from 20 patients who underwent surgery for gastric cancer. According to UICC TNM system, 3 of the cases were Ia, 2 cases II, 4 cases IIIa, 5 cases IIIb, and 6 cases IV. Results: Immunohistochemical staining revealed eight cases as negative (less than $10\%$), twelve cases as postive (more than $10\%$). The locations of mutations were as follows; 7 cases had point mutation at exon 4, and 3 cases point mutation at exon 8. There was no mutation at exon 5, 6, 7 and 9. The mutation was observed in 1 case out of 8 p53 oncoprotein negative cases, and 7 cases out of 12 p53 positive cases. The mutation was more common in p53 positive cases (P<0.05), However, there was no significant correlation between p53 mutation observed by DNA sequencing after laser capture microdissection and expression of p53 oncoprotein. Conclusion: These result suggest that he expression of p53 oncoprotein not to be related to the mutation of p53 gene at exons 4 through 9 in gastric cancer.

• BMB Reports
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• v.39 no.5
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• pp.469-478
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• 2006

Vascular endothelial growth factor (VEGF)-A, a major regulator for angiogenesis, binds and activates two tyrosine kinase receptors, VEGFR1 (Flt-1) and VEGFR2 (KDR/Flk-1). These receptors regulate physiological as well as pathological angiogenesis. VEGFR2 has strong tyrosine kinase activity, and transduces the major signals for angiogenesis. However, unlike other representative tyrosine kinase receptors which use the Ras pathway, VEGFR2 mostly uses the Phospholipase-$C{\gamma}$-Protein kinase-C pathway to activate MAP-kinase and DNA synthesis. VEGFR2 is a direct signal transducer for pathological angiogenesis including cancer and diabetic retinopathy, thus, VEGFR2 itself and the signaling appear to be critical targets for the suppression of these diseases. VEGFR1 plays dual role, a negative role in angiogenesis in the embryo most likely by trapping VEGF-A, and a positive role in adulthood in a tyrosine kinase-dependent manner. VEGFR1 is expressed not only in endothelial cells but also in macrophage-lineage cells, and promotes tumor growth, metastasis, and inflammation. Furthermore, a soluble form of VEGFR1 was found to be present at abnormally high levels in the serum of preeclampsia patients, and induces proteinurea and renal dysfunction. Therefore, VEGFR1 is also an important target in the treatment of human diseases. Recently, the VEGFR2-specific ligand VEGF-E (Orf-VEGF) was extensively characterized. Interestingly, the activation of VEGFR2 via VEGF-E in vivo results in a strong angiogenic response in mice with minor side effects such as inflammation compared with VEGF-A, suggesting VEGF-E to be a novel material for pro-angiogenic therapy.

• BMB Reports
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• v.44 no.7
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• pp.462-467
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• 2011

Up-regulation of selected matrix metalloproteinases (MMPs) such as MMP-9 contributes to inflammatory processes during the development of various skin diseases, such as atopic dermatitis. In this study, we examined the effect of a cell-permeable superoxide dismutase (Tat-SOD) on TNF-${\alpha}$-induced MMP-9 expression in human keratinocyte cells (HaCaT). When Tat-SOD was added to the culture medium of HaCaT cells, it rapidly entered the cells in dose- and time-dependent manners. Tat-SOD decreased TNF-${\alpha}$-induced reactive oxygen species (ROS) generation. Tat-SOD also inhibited TNF-${\alpha}$-induced NF-${\kappa}B$ DNA binding activity. Treatment of HaCaT cells with Tat-SOD significantly inhibited TNF-${\alpha}$-induced mRNA and protein expression of MMP-9, as measured by RT-PCR and Western blot analysis. In addition, Tat-SOD suppressed TNF-${\alpha}$-induced gelatinolytic activity of MMP-9. Taken together, our results indicate that Tat-SOD can suppress TNF-${\alpha}$-induced MMP-9 expression via ROS-NF-${\kappa}B$-dependent mechanisms in keratinocytes, and therefore can be used as an immunomodulatory agent against inflammatory skin diseases related to oxidative stress.

• Journal of Physiology & Pathology in Korean Medicine
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• v.17 no.3
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• pp.819-825
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• 2003

pharbitis nil and Taraxacum mongolicum are representative herbs that have been used for cancer treatment in Korean traditional medicine. To understand the molecular basis of the antitumor function, we analyzed the effect of these herbs on proliferation and apoptosis of tumor cells using a gastric cancer cell line AGS. Cell counting assay showed that pharbitis nil strongly inhibit cell proliferation Of AGS whereas Taraxacum mongolicum exhibit no detectable effect on cellular growth. [³H]thymidine uptake analysis also demonstrated that DNA replication of AGS is suppressed in a dose-dependent manner by treatment with pharbitis nil. Additionally, tryphan blue exclusion assay showed that Pharbitis nil induce apoptotic cell death of AGS in a dose-dependent. To explore whether anti antiproliferative and/or proapototic property of Pharbitis nil is associated with their effect on gene expression, we performed RT-PCR analysis of cell cycle- and apoptosis-related genes. Interestingly, mRNA expression levels of c-Jun, c-Fos, c-Myc, and Cyclin D1 were markedly reduced by Pharbitis nil. Taraxacum mongolicum also showed inhibitory action on expression of these growth-promoting protooncogene but there effects are less significant, as compared to Pharbitis nil. Furthermore, it was also found that Pharbitis nil activates expression of the p53 tumor suppressor and its downstream effector p21Waf1, which induce G1 cell cycle arrest and apoptosis. Collectively, our data demonstrate that Pharbitis nil induce growth inhibition and apoptosis of human gastric cancer cells and these effects are accompanied with down-and up-regulation of growth-regulating protooncogenes and tumor suppressor genes, respectively. This observation thus suggests that the anticancer effect of Pharbitis nil might be associated with its regulatory capability of tumor-related gene expression.

• Parasites, Hosts and Diseases
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• v.44 no.3
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• pp.187-196
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• 2006

The mammalian trematode Paragonimus westermani is a typical digenetic parasite, which can cause paragonimiasis in humans. Host tissues and blood cells are important sources of nutrients for development, growth and reproduction of P. westermani. In this study, a cDNA clone encoding a 47 kDa hemoglobinase of P. westermani was characterized by sequencing analysis, and its localization was investigated immunohistochemically. The phylogenetic tree prepared based on the hemoglobinase gene showed high homology with hemoglobinases of Fasciola hepatica and Schistosoma spp. Moreover, recombinant P. westermani hemoglobinase degradaded human hemoglobin at acidic pH (from 3.0 to 5.5) and its activity was almost completely inhibited by E-64, a cysteine proteinase inhibitor. Immunohistochemical studies showed that P. westermani hemoglobinase was localized in the epithelium of the adult worm intestine implying that the protein has a specific function. These observations suggest that hemoglobinase may act as a digestive enzyme for acquisition of nutrients from host hemoglobin. Further investigations may provide insights into hemoglobin catabolism in P. westermani.

• Biomolecules & Therapeutics
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• v.25 no.4
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• pp.396-403
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• 2017

Under normal, non-stressed conditions, intracellular p53 is continually ubiquitinated by MDM2 and targeted for degradation. However, in response to severe genotoxic stress, p53 protein levels are markedly increased and apoptotic cell death is triggered. Inhibiting the ubiquitination of p53 under conditions where DNA damage has occurred is therefore crucial for preventing the development of cancer, because if cells with severely damaged genomes are not removed from the population, uncontrolled growth can result. However, questions remain about the cellular mechanisms underlying the regulation of p53 stability. In this study, we show that p53-inducible gene 3 (PIG3), which is a transcriptional target of p53, regulates p53 stability. Overexpression of PIG3 stabilized both endogenous and transfected wild-type p53, whereas a knockdown of PIG3 lead to a reduction in both endogenous and UV-induced p53 levels in p53-proficient human cancer cells. Using both in vivo and in vitro ubiquitination assays, we found that PIG3 suppressed both ubiquitination- and MDM2-dependent proteasomal degradation of p53. Notably, we demonstrate that PIG3 interacts directly with MDM2 and promoted MDM2 ubiquitination. Moreover, elimination of endogenous PIG3 in p53-proficient HCT116 cells decreased p53 phosphorylation in response to UV irradiation. These results suggest an important role for PIG3 in regulating intracellular p53 levels through the inhibition of p53 ubiquitination.