• Journal of Oral Medicine and Pain
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• v.34 no.3
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• pp.261-276
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• 2009

Lactacystin, a microbial natural product synthesized by Streptomyces, has been commonly used as a selective proteasome inhibitor in many studies. Proteasome inhibitors is known to be preventing the proliferation of cancer cells in vivo as well as in vitro. Furthermore, proteasome inhibitors, as single or combined with other anticancer agents, are suggested as a new class of potential anticancer agents. This study was undertaken to examine in vitro effects of cytotoxicity and growth inhibition, and the molecular mechanism underlying induction of apoptosis in SCC25 human tongue sqaumous cell carcinoma cell line treated with lactacystin. The viability of SCC25 cells, human normal keratinocytes (HaCaT cells) and human gingiva fibroblasts (HGF-1 cells), and the growth inhibition of SCC25 cells were assessed by MTT assay and clonogenic assay respectively. The hoechst staining, hemacolor staining and TUNEL staining were conducted to observe SCC25 cells undergoing apoptosis. SCC25 cells were treated with lactacystin, and Western blotting, immunocytochemistry, confocal microscopy, FAScan flow cytometry, MMP activity, and proteasome activity were performed. Lactacystin treatment of SCC25 cells resulted in a time- and does-dependent decrease of cell viability and a does-dependent inhibition of cell growth, and induced apoptotic cell death. Interestingly, lactacytin remarkably revealed cytotoxicity in SCC25 cells but not normal cells. And tested SCC25 cells showed several lines of apoptotic manifestation such as nuclear condensation, DNA fragmentation, the reduction of MMP and proteasome activity, the decrease of DNA contents, the release of cytochrome c into cytosol, the translocation of AIF and DFF40 (CAD) onto nuclei, the up-regulation of Bax, and the activation of caspase-7, caspase-3, PARP, lamin A/C and DFF45 (ICAD). Flow cytometric analysis revealed that lactacystin resulted in G1 arrest in cell cycle progression which was associated with up-regulation in the protein expression of CDK inhibitors, $p21^{WAF1/CIP1}$ and $p27^{KIP1}$. We presented data indicating that lactacystin induces G1 cell cycle arrest and apoptois via proteasome, mitochondria and caspase pathway in SCC25 cells. Therefore our data provide the possibility that lactacystin could be as a novel therapeutic strategy for human tongue squamous cell carcinoma.

• The korean journal of orthodontics
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• v.31 no.5 s.88
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• pp.525-534
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• 2001

Bone remodeling in response to force requires coordinated actions of osteoblasts, osteoclasts, osteocytes, and periodontal ligament cells. Coordination among these cells may be mediated, in part, by cell-to-cell communication via gap junctions. This study was designed to evaluate the expression of gap junction, connection 43 In periodontal tissue during the experimental movement of rat's incisors, by LSAB(labelled streptavidine biotin) immunohistochemical staining for connexin 43. Twenty seven Sprague-Dawley rats were divided into a control group(3 rats), and 6 experimental groups(24 rats) where 75g of force was applied from helical springs across the maxillary incisors. Rats of experimental groups were sacrificed at 12 hours, 1, 4, 7, 14 and 28 days after force application, respectively. And the tissues of a control group and experimental groups were studied immunohistochemically. The results were as follows : 1. In control group, the expression of connexin 43 was rare in gingiva, dentin, cementum, periodontal ligament, and bone cells. 2. In experimental group, the expression of connexin 43 was increased in pulp, periodontal ligament, osteoblasts, and osteoclasts, comparing to that in control. And it was rare in gingiva, dentin, and odontoblasts regardless of the duration of force application, which was not different from that of control group. 3. The expression of connexin 43 in pulp of experimental group began to increase in 4-day after force application and got to the highest degree at 7-day. And it decreased after 14-day to be similar to that of control group at 28-day. 4. The expression of connexin 43 in periodontal ligament was noted in small capillaries adjacent to alveolar bone, showing higher intensity of immunolabelling after 4-day And it was stronger in the pressure side than in tension side of periodontal ligament. After 7-day, decrease in connexin 43 expression was observed. 5. The expression of connexin 43 in alveolar bone began to increase 1-day, reached to the highest degree at 4-day, and decreased at 7-day. And the expression in osteoclasts was more than that in osteoblasts or osteocyte at 7-day.

• Journal of Oral Medicine and Pain
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• v.24 no.4
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• pp.361-374
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• 1999

F. nucleatum is a gram-negative obligate anaerobe which is the principal and most frequent cause of gingival inflammation and is the predominant pathogen isolated in subsequent periodontal breakdown. It is also one of the most numerous bacteria found in subgingival plaque samples from healthy sites; its numbers are about 10-fold greater in plaque from periodontally diseased sites. The purpose of this study is to examine the effects of outer membrane(OM), outer membrane vesicle(OMV), and lipopolysaccharide(LPS) from F. nucleatum ATCC 25586 strain on the growth of human gingival fibroblasts and HOS 941 cells, and on the $TNF-{\alpha}$ production / $TNF-{\alpha}$ mRNA expression of mouse splenocytes. For the examination of cytotoxic effects, $TNF-{\alpha}$ production and $TNF-{\alpha}$ mRNA expression, the MTT assay, the ELISA and the RT-PCR were performed, respectively. All extracts of F. nucleatum tested were cytotoxic to both of human gingival fibroblasts and HOS 941 cells, and the significant difference of cytotoxic activity among the extracts was not observed. In the effects of these extracts on the $TNF-{\alpha}$ production / $TNF-{\alpha}$ mRNA expression of mouse splenocytes, all extracts of F. nucleatum tested also stimulated the $TNF-{\alpha}$ production / $TNF-{\alpha}$ mRNA expression, but the effects of the OM extracts on the $TNF-{\alpha}$ production / $TNF-{\alpha}$ mRNA expression were higher than those of the OMV and the LPS extracts. The pattern of the $TNF-{\alpha}$ mRNA expression was similar to that of the $TNF-{\alpha}$ production. These results indicate that F. nucleatum seems to contribute to the pathogenesis of periodontal diseases at least by its cytotoxicity, directly and its $TNF-{\alpha}$ production, indirectly.

• Journal of Periodontal and Implant Science
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• v.29 no.4
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• pp.803-823
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• 1999

In order to reveal immunopathogenesis of periodontal tissue destruction, it is important to clarify the molecular mechanism of trafficking and retention of activated leukocytes, including monocytes/macrophages. Gingival fibroblasts may be involved in the regulation of inflammatory cell accumulation in the extravascular periodontal connective tissues via cytokine production and surface expression of adhesion molecules. In this study, it was investigated the molecular basis for the adhesive interactions between monocytes and fibroblasts such as peri-odontal ligament fibroblast(PDLF), human gingival fibroblast(HGF), and human dermal fibroblast(HDF). First, it was examined the evidence whether monocyte-fibroblast cell contact may cause signal transduction in fibroblasts. Being directly in contact with fixed human monocyte cell line THP-1, or U937, upregulation of IL-6 production, $TNF-{\alpha}$ mRNA expression and increased cell proliferation could be seen for fibroblasts. IL-6 production induced by monocyte- fibroblast coculture were further increased when fibroblasts had been pretreated with $IFN-{\gamma}$ or $IL-1{\beta}$ , and monocytes with LPS. Next, it was examined the expression of ICAM-1 which has been known to be involved in accumulation and activation of leukocytes in inflammatory diseases such as periodontitis. ICAM-1 was upregulated up to 10-fold on PDLF, HGF, and HDF by exposure to $IFN-{\gamma}$ or $IL-1{\beta}$. Furthermore, anti-ICAM-1 monoclonal antibody clearly blocked cocultureinduced IL-6 production by fibroblasts, suggesting that $ICAM-1/{\beta}_2$integrin pathway is involved in periodontal fibroblastmonocyte interaction. Overall, these findings provide evidence that periodontal fibroblasts could be involved in the accumulation and retention of monocytes/macrophages in periodontal inflammatory lesion at least in part by ICAM-1 expression. In addition, periodontal fibroblast-monocyte interaction could cause activation signals in fibroblasts intracellularly which result in cytokine production and cell proliferation. Thus, periodontal fibroblasts are speculated to play an important role in immunoregulation and tissue destruction in chronic periodontal diseases by interaction with monocytes/macrophages.

• Korean Journal of Food Science and Technology
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• v.33 no.6
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• pp.753-759
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• 2001

To evaluate antiinflammation of Aloe vera peel, antiimflammation substances were extracted from Aloe vera peel and identified, and we investigated the effect of the its substance the inhibitory effect on the activity of hyaluoronidase, elastase, collagenase and prostaglandin endoperoxide synthase. The water extract from Aloe vera peel were successfully purified with solvent fractionation, silica gel column chromatography, preparative thin layer chromatography and UV spectrometer. Two purified active substances were identified as aloe-emodin and barbaloin by Mass Spectrometer, $^1H-NMR$ and FT-IR. Aloe-emodin and barbaloin. $IC_{50}$ values of aloe-emodin and barbaloin against hyaluronidase activity were 40 and $70\;{\mu}g/mL$, respectively. Leuckocyte elastase, which is related to the destruction of various tissue, $IC_{50}$ values of them were 50 and $60\;{\mu}g/mL$, respectively. $IC_{50}$ values of aloe-emodin and barbaloin against collagenase activity were 40 and $60\;{\mu}g/mL$, respectively. and $IC_{50}$ values of aloe-emodin and barbaloin aganist the prostaglandin endoperoxide synthase, which play an important role in inflammatory reactions, were 40 and $70\;{\mu}g/mL$, respectively. Inhibitory effects of aloe-emodin, barbaloin and aspirin against carrageenan paw edema were 74.9, 52.9 and 51.9% as inhibiton percentage, respectively, at dose of 100 mg/kg and that of indomethancin was 49.7 at dose of 10 mg/kg. Cell cytotoxicity of barbaloin against human gingival cells was lower than that of aloe-emodin. Aloe-emodin and barbaloin did not show cytotoxicity against human gingival cells at concentration of 1.0 and $5.0\;{\mu}g/mL$, However, aloe-emodin and barbaloin showed less cytotoxicity than chlorhexidine, which usually have been used as the agent of anticaries and antiinflammation. These results suggested that aloe-emodin and barbaloin from Aloe vera peel have the effect of anticaries and antiinflammation.

• Journal of Periodontal and Implant Science
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• v.25 no.1
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• pp.133-145
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• 1995

The migration and proliferation of periodontal ligament cells are desired goal of periodontal regeneration therapy. PDGF and $TGF-{\beta}1$ are well known to regulate the cell activity of mesenchymal origin cell. The purpose of this study was to determine the effects of these growth factors on human gingival fibroblast and periodontal ligament cell actvity, and to identify the regulatory effect of $TGF-{\beta}1$ on the response to PDGF by MIT assay. Human gingival fibroblast and periodontal ligament cells were cultured from extracted teeth for non-periodontal reason. Cultured human gingival fibroblast and periodontal ligament cells in vitro were treated with polyperpetide growth factor PDGF and $TGF-{\beta}1$ in both a dose and time - dependent manner. Cell morphology were determined by inverted microscope and cell acitivity were determined by MIT assay. The result of this study demonstrated that PDGF and $TGF-{\beta}1$ were not changed the morphology of these cell compared with control group. PDGF or $TGF-{\beta}1$ increased cell activity of periodontal ligament cell in dose and time dependent manner but gingival fibroblast were decreased to the level of control group at third day. Additionally, incubation with $TGF-{\beta}1$ addition to PDGF resulted in a enhanced cell activity of PDGF. Therefore, cell acitivty of gingival fibroblast were not changed compared with control group. This stiudy demonstrates that PDGF and $TGF-{\beta}1$ are major mitogens for human periodontal ligament cell in vitro, and $TGF-{\beta}1$ is a regulator of cell activity to PDGF in human gingival fibroblast and periodontal ligament cell.

• Journal of Periodontal and Implant Science
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• v.29 no.1
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• pp.31-40
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• 1999

Many factors may affect periodontal changes during the physiologic conditions of woman(e.g. puberty, menstrual cycle, pregnancy, menopause). Recently many research has focused on the immunological changes of host, but the exact mechanism is not clear. Collagen is a major constituent of periodontium, and collagenase specifically digests the collagen and plays a role in destruction of periodontal tissue. So, I suppose that it participates with the cytokines in the inflammation of gingiva and vascular response during the changes of female sex hormones. Because there are some evidences of the existence of the receptors of estrogen and progesterone in the gingiva, it may be a target tissue of female sex hormones. In this experiment, gingival fibroblast and periodontal ligament cell were cultured in the presence of various concentrations of estrogen or progesterone corresponding to the menstrual cycle and pregnancy. Collagenase activity of the supernatant of culture media was determined by Spectrophotometric collagenase assay. The enzyme activity was calculated by the % decrease of the coated collagen. 1. The estrogen at both concentrations had no effect on the activity of collagenase of the gingival fibroblast. 2. The progesterone had some effect on the collagenase activity of the gingival fibroblast at low and high concentration of menstrual cycle, and elevated the enzyme activity at all range of pregnancy concentrations. 3. In periodontal ligament cells, estrogen elevated the enzyme activity at the early pregnancy concentration and progesterone elevated at the concentration just before menstruation. In this experiment, pregesterone elevated the collagenase activity of gingival fibroblast and periodontal ligament cells. But the mechanism of the up-regulation of the enzyme activity was not confirmed. The more experiments of direct effect of progesterone on gingival at the molecular level(e.g. northern blot analysis) can reveal the exact mechanism.

• Journal of Oral Medicine and Pain
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• v.21 no.1
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• pp.153-171
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• 1996

저수준레이저를 이용하여 창상이나 병소의 치유과정에 대한 효과를 조사하기 위하여 많은 연구가 시행되었다. 연구에 의하면 갈륨비소 레이저광이 생체자극효과를 가진다고 하며, 저수준레이저를 조사하면 단백질과 핵산 (DNA) 합성을 자극하여 치은섬유아세포의 증식을 촉진한다고 보고하였다. 외상병소나 근육병소의 치료에 사용된 레이저치료법에 관한 관심이 점증함에 따라 저수준레이저요법 (LLLI)의 치유효과를 설명하기 위하여 분자생물학적 수준의 연구를 시행하기에 이르렀다. 보고에 의하면 Mutans Streptococcide 는 LLLI를 사용시 증식이 촉진되며, 다른 세균에서도 유사한 증식효과가 나타날 것이라고 주장하였다. 그러므로 LLLI가 피부감염을 야기하는 가장 흔한 원인인 Staphylococcus aureus 도 마찬가지로 증식이 촉진되는 지를 조사해볼 필요가 있으며, 또한 감염과 같이 특정 병적 상태에서의 저수준레이저광의 효과는 아직까지 명확하게 밝혀져 있지 않았다. 그러므로 본 연구의 목적은 첫째, Staphyloc occus aureus 의 증식에 대한 저수준레이저광의 효과를 조사하는 실험이며, 둘째 Staphylococcus aureus 로 가염된 피부창상에 대한 저수준레이저광의 효과를 판정하는데 있다. 34개의 Staphylococcus aureus 배양표본을 사용하여 48시간의 세포주기동안 조사기간과 조사시간, 그리고 레이저 펄스(laser pulse)형에 따라 3가지 실험을 시행하여 증식에 가장 효과적인 상태와 가장 비효과적인 상태의 갈륨비소 반도체 레이저펄스를 결정하였다. 이후 지름 약 6 mm의 개방창상을 44마리 백서의 양측 대퇴부에 형성하여 모든 창상에 S. aureus를 감염시켰다. 모든 표본은 펄스형과 조사방법 (중앙조사법과 주변조사법)에 따르는 실험을 하기 위하여 4가지로 분류하였다. 각 백서의 양측 창상중 하나는 1,3,5,7일 마다 각 실험의 방법에 따라 레이저를 조사하고 실험동물의 다른 창상은 대조군으로서 사용하였다. 모든 창상의 면적은 실험 1,3,5,7 일째에 일정한 거리에서 사진촬영하여 면적계를 이용, 측정한 후 통계적인 의의를 조사하였다. 본 연구의 결과는 저수준레이저는 특정 조건하에서 S. aureus의 증식을 촉진하였다. 그러나 S. aureus에 감염된 창상을 저수준레이저로 조사시 치유가 촉진되었다. 중앙 조사법고 주변조사법에 의한 창상치유효과는 통계적인 의의가 보이지 않았다. 따라서 결론적으로 S. aureus 에 감염된 창상에 직접 또는 간접적이든 pulse의 종류에 관계없이 조사하는 경우 치유효과가 나타나는 것은 정사주위 조직의 LLLI 자극효과가 염증의 확산을 억제한다고 말할수 있다.

• Journal of Korean Academy of Oral and Maxillofacial Radiology
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• v.27 no.1
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• pp.107-122
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• 1997

Radiation sensitivity data was generated for two human cancer cell lines(KB, RPMI 2650) and human primary gingival fibroblast was tested three times using a viable cell number counting with a hemocytometer, MTT(3-[4,5-Dimethylthiazol2-yl]-2,5-diphenyl tetrazolium bromide) assay, and LDH(Lactate dehydrogenase) assay. Single irradiation of 2, 4, 6, 10, 15, 20Gy were applied to the tumor cell lines and the primary cultured gingival fibroblast The two fractions of 4Gy and 10Gy were seperated with a 4 hour time interval. The irradiation was done with 241.5cGy/min dose rate using /sup 137/Cs MK cell irradiator at room temperature. The obtained results were as followed : 1. There was significantly different viable cell numbers as the amount of radiation dose on the tested cells were cell number counted with a hemocytometer. In fractions, there were more viable cells remaining. 2. Phase-contrast microscopically, radiation-induced morphologic changes were pronounced on the tumor cells, however, almost no differences on the gingival fibroblast. 3. There was significantly different absorbance at 2Gy on RPMI 2600, 4Gy on KB and GF in MTT assay. In fractions, the absorbance was significantly higher on KB. 4. The level of extracellular LDH activity in the experimental group was significantly higher in the 2-4Gy than the control group. 5. The total level of extracellular and intracellular LDH activity was decreased as increased amounts of radiation dose was applied.

• Journal of Periodontal and Implant Science
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• v.31 no.1
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• pp.135-148
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• 2001

Gingival fibroblasts are major cellular component of gingiva. However, the molecular mechanisms of senescence of human gingival fibroblasts are unknown. Human fibroblasts undergo replicative senescence in vitro after a limited number of population doublings. A reduced rate of proliferation is a prominent phenomenon observed in senescent fibroblasts. This phenomenon is controled by cell cycle regulatory proteins. The purpose of present study was to investigate the effect of replicative senescence on cell cycle progression and to find out its molecular mechanisms in human gingival fibroblasts. Replicative senescence of gingival fibroblasts were induced by subsequent cultures that were repeated up to 18 passage. In the present study, I examined change of cell proliferation, cell activity, cell viability and cell cycle progression during the replicative process. Also, I examined expression of cell cycle regulatory proteins which was estimated by western blot analysis. Cell proliferation, cell activity and cell viability of gingival fibroblasts were notably decreased with increase of population doubling level(PDL). S phase was decreased and G1 phase was increased with increase of PDL. Western blot analysis showed that levels of P16, p21 and p53 of senescent gingival fibroblasts(PDL41, PDL58) were higher than young fibroblasts(PDL27) and cdk4 were lower than young fibroblasts(PDL27). In conclusion, these results suggest that proliferative function of human gingival fibroblasts may be decreased by replicative senescence and its molecular mechanisms may be activatied with p16, p21, p53 and pRB, and repressed wtih cdk4.