Recently chronic inflammation is focused on the association with cancer progression and acquisition of aggressive biologic behaviors, such as invasion, metastasis, and resistance to chemotherapeutic reagents. Due to the close vicinity within oral cavity, oral cancer may be intimately associated with chronic periodontitis. The present study was done to observe the effect of chronic periodontitis on oral cancer cells by utilizing P. gingivalis infection, a major pathogen in chronic periodontitis. We analyzed and compared the mRNA expression levels of epithelial-mesenchymal transition (EMT) markers in non-infected and P. gingivalis-infected oral cancer cells. Eighty-six genes, which are well known as EMT markers, were analyzed using commercially available EMT microarray plates, performed in triplicate. Among the 86 genes, the expression of 26 was increased (≥ 2 fold) by P. gingivalis, whereas that of 7 genes was decreased (≥ 2 fold). Our study suggests that P. gingivalis infection evokes significant changes in EMT-related genes. Further observations on molecular mechanisms underlying these changes may help to clarify the role of chronic periodontitis on cancer progression and to develop more efficient preventive and therapeutic modalities for oral cancer. (182 words)

목적: 항균제 polyhexamethylene Biguanide(PHMB), epigallocatechin gallate(EGCG) 및 PHMB/EGCG 혼합물의 각막상피세포(primary human corneal epithelial cells, HCEpiCs)에 대한 급성독성을 평가하고자 하였다. 방법: 각막상피세포를 0.00001~0.005% PHMB, 0.001~5% EGCG 및 0.00005% PHMB/0.05% EGCG 혼합물이 각각 포함된 배양액에서 30분, 60분, 120분 및 240분 동안 배양하였다. 배양한 각막상피세포를 고정한 다음 Draq 5로 염색하고 공초점현미경과 ImageXpress UltraTM를 이용하여 세포형태를 관찰하여 세포 생존율과 세포자살(apoptosis)을 비교하였다. 결과: 배양된 각막상피세포는 0.00005% 이하의 PHMB 농도 및 0.05% 이하의 EGCG 농도에서는 세포 독성이 나타나지 않았다. 0.00005% PHMB/0.05% EGCG 혼합물이 포함된 배양액에서 급성 세포독성은 관찰되지 않았으나 240분 배양시킨 경우에는 손상된 각막상피세포 수가 증가하고 생존 세포의 수는 감소하였다. 결론: 항균 시너지 효과를 갖는다고 보고된 0.00005% PHMB/0.05% EGCG 혼합물의 경우 각막상피세포에 대한 급성독성은 없었으나 만성효과에 대해서는 추가적인 연구가 필요할 것으로 생각된다.

Oral squamous cell carcinoma (OSCC) is the most common type of oral malignancy. Numerous therapies have been proposed for its cure. Research is continually being conducted to develop new forms of treatment as current therapies are associated with numerous side-effects. Luteolin, a common dietary flavonoid, has been demonstrated to possess strong anti-cancer activity against various human cancer cell lines. Nevertheless, research into luteolin-based anticancer activity against oral cancer remains scarce. Thus, the objective of this study was to assess the effect of luteolin as an anti-cancer agent. After treatment with luteolin, Ca9-22 and CAL-27 oral cancer cells showed condensed nuclei and enhanced apoptotic rate with evidence of mitochondria-mediated apoptosis. Epithelial-mesenchymal transition (EMT) is closely related to tumor migration and invasion. Luteolin suppressed cancer cell invasion and migration in the current study. Elevated expression of E-cadherin, an adherens junction protein, was evident in both cell lines after luteolin treatment. Luteolin also significantly inhibited transcription factors (i.e., N-cadherin, Slug, Snail, Twist, and ZEB-1) that regulated expression of tumor suppressors such as E-cadherin based on Western blot analysis and quantitative PCR. Thus, luteolin could induce mitochondrial apoptosis and inhibit cancer cell invasion and migration by suppressing EMT-induced transcription factors.

This study examined the motility of either the unattached(upper) or attached(lower) Hanwoo sperm to bovine oviduct epithelial cell(BOEC) monolayers to determine whether there are any changes in their motility during co-culture. The cleavage and blastocyst development rate were compared among different preincubation methods in-vitro, after oocytes were fertilized in-vitro with Hanwoo sperm on BOEC monolayers. The motility of frozen-thawed sperm in BOEC co-culture group was significantly higher than controls, especially at 5 hours and 6 hours (p<0.05) of incubation, in sperm treatment medium without heparin and caffeine. The motility of frozen-thawed sperm in BOEC co-culture group was significantly higher than controls, especially at 3 hours (p<0.05) and 6 hours (p<0.01), in sperm treatment medium containing heparin and caffeine. The motility of the attached( lower) sperm was significantly higher than the unattached(upper) sperm during co-culture with BOEC at all times(p<0.01 or p<0.05), except for 6 hours. After Hanwoo oocytes were fertilized in-vitro with the sperm that had been co-cultured with BOEC in sperm treatment medium containing heparin and caffeine, we determined the cleavage and blastocyst development rate, according to the preincubation methods. Both the cleavage and blastocyst development rate from 2 hour preincubation group were the highest, but significant difference was not recognized. These results show that BOEC plays an important role on sperm hyperactivation related to capacitation regardless of heparin and caffeine in sperm treatment medium. However, oviduct epithelial cell had no significant effect on the development of embryos after in-vitro fertilization in the presence of added heparin and caffeine in sperm treatment medium.

Expression of epithelial cell adhesion molecule (EpCAM) in the early phase of hepatocarcinogenesis induced by diethylnitrosamine (DEN) was investigated. At 14 days of age, 60 ICR mice were divided into two groups and treated with saline (group 1) or DEN (group 2, 10 mg/kg of body weight, i.p. injection), and were sacrificed at 6 h and 1, 2, 3, 7, and 28 days after treatment with saline or DEN. During necropsy, half of the liver from saline- or DEN-treated mice was processed for histopathological examination and immunohistochemical staining of EpCAM and apoptosis. The remaining liver tissue was snap-frozen in liquid nitrogen for RNA extraction and analysis of EpCAM mRNA expression. Immunohistochemical examination showed that EpCAM expression was detected only in a small number of hepatocytes from saline-treated mice and its expression was detected in bile duct cells and round cells around portal areas, as well as hepatocytes in the livers of DEN-treated mice. In addition, multiple apoptotic cells were found in the livers of mice treated with DEN. EpCAM mRNA expression was significantly higher in DEN-treated mice at 1, 7, and 28 days compared to saline-treated mice at 6 h (P<0.01). Taken together, EpCAM expression and apoptosis were increased in liver by DEN treatment.

Malignant tumor cells outgrow new blood vessel formation and tend to be in hypoxic state. Hypoxic cancer cells adapt to hypoxic conditions by transforming its characteristics. On the other hand, one of the most important features of cancer cells is that carcinoma cells loses its inherent epithelial phenotype and acquires mesenchymal characteristics, called as epithelial-mesenchymal transition(EMT). It has been already well known that EMT contributes to tumor invasion and metastasis. The present study investigated whether hypoxia play a major role in induction of phenotypic changes of oral squamous cell carcinoma(OSCC). Furthermore, the mechanism of EMT in oral squamous cell carcinoma cells by hypoxia has been clarified. To mimic hypoxic condition, cobalt chloride and desferoxamine, well-known hypoxic mimetic agents, were used. This study shows that hypoxia suppresses the expression of E-cadherin(epithelial marker) and increases vimentin and N-cadherin( mesenchymal markers) in OSCC. In addition, α5 integrin protein, which is a receptor for fibronectin and an important molecule for tumor invasion, is prominently induced by hypoxia.

Epigallocatechin-3-gallate (EGCG) and theaflavins (TF) are polyphenols included in green and black teas, respectively. Both green and black teas have been studied for their potential health benefits for cancer. Hypoxia-inducible factor (HIF) has been implicated multiple physiological and pathophysiological pathways, particularly, oncogenesis. But, the molecular pathways that govern the cell response to EGCG are not fully elucidated. The present study investigated the intracellular mechanism in oral squamous cell carcinoma (OSCC) cells treated with EGCG, focusing on HIF-1 expression and its effect on epithelial phenotype. EGCG decreased phosphorylated Raf-1 protein in YD 8 OSCC cell, but B-raf protein was not affected at all by EGCG and TF. In addition, we here found that EGCG regulated HIF-1α expression independent of Raf-1 protein. Taken together with our previous result, the result imply that EGCG is attributed to the HIF-1α expression via Raf/MEK/ERK pathway, and the HIF-1α expression is associated with the change of epithelial phenotype in OSCC cell.

The tumor suppressor gene, phosphate and tensin homologue(PTEN) has been shown to dephosphorylate the phosphatidylinositol 3-kinase(PI 3-K)-generated phosphatidylinositol(3-5)-triphosphate in vivo, thus interfering with the potentially oncogenic signals emanating from PI 3-K. Promoter hypermethylation of CpG islands has recently been shown to be an epigenetic change resulting in loss of function in some genes involved in cell cycle regulation and DNA repair. Immunohistochemal staining for monoclonal antibody 6H2.1 was performed from paraffin embedded blocks of 20 benign epithelial lesions and 40 head and neck squamous cell carcinomas(HNSCCs). Immunoreactivity was graded semiquantitatively by considering the percentage and intensity of the staining of the tumor cells. Also, this study tried to identify PTEN methylation in benign epithelial lesions(24 cases) and HNSCCs(44 cases of paraffin embedded blocks, 4 cases of frozen tissues) using methylation-specific PCR(MSP). In HNSCCs, immunoreactive scores of stage 1 and 2(12 cases, average score 85.2) were higher than those of stage 3 and 4(15 cases, 41.9) and statistically significant(P=0.017). Immunoreactive scores of moderate and poorly differentiated carcinomas(22 cases, 61.6) are more or less lower than those of well differentiated carcinoma(15 cases, 87.0) but not significant(P=0.361). Among 24 cases of benign epithelial lesions, 12 cases showed unmethylated PTEN but none methylated. In HNSCCs, 22 of 44 paraffin embedded blocks showed unmethylated PTEN but none methylated, and all 4 frozen tissue revealed unmethylated PTEN, one of which(25%) methylated. We consider that the loss of PTEN protein expression may be associated with the progression of HNSCCs and the other alteration rather than methylation may be important in the inactivation of PTEN in HNSCCs.

Expression of invasion/metastasis suppressor, E-cadherin, is reduced in many types of human carcinomas. Although somatic and germline mutations in the CDH1, which encodes the human E-cadherin, have frequently been reported in cases with diffuse gastric and lobular breast cancers, irreversible genetic inactivations are rare in other human carcinomas. Recently, it has been well documented that some genes in human cancers may be inactivated by altered CpG methylation. Herein, we determined the expression and methylation status of E-cadherin in oral squamous cell carcinoma(SCC) by immunohistochemistry and methylation-specific PCR. The expression of E-cadherin was significantly higher in the well-differentiated oral SCCs than the moderately or poorly differentiated ones. None of eight tested benign epithelial hyperplasias showed aberrant methylation, whereas five of 12 oral squamous cell carcinomas showed aberrant methylation. When we compared E-cadherin expression with methylation status, oral SCCs with normal methylation showed a higher expression of E-cadherin than those with methylation. These findings suggest that aberrant CpG methylation of CDH1 promoter region is closely associated with transcriptional inactivation and might be involved in tumor progression of the oral mucosa.

Porcine oviduct epithelial cells (POEC) are widely used in co-culture experiments to improve early embryonic development, in vitro fertilization in embryo transfer programs for domestic animals and in vitro maturation of immature germ cells. POEC were mechanically isolated and cultured in tissue culture medium 199. Cells grew continuously, and confluent monolayers were formed after 7 days. After forming confluent monolayer of epithelial cells, supernatant was collected as the condition medium for maturing round spermatids in vitro. Round spermatids were also separated mechanically and cultured in the POEC condition medium. In this study we observed that 20% of round spermatid cultured were matured into elongating spermatid after 24 h, and about 10% of round spermatid cultured showed complete elongation (elongated spermatid) within 24~48 h of in vitro culture. No further development was observed within 50~72 h and transformed cells lost their viability after 72 h. These preliminary findings suggest that the condition medium from POEC may be possible to overcome the round spermatid block by improving the milieu of culture system.

본 연구는 VSV-G glycoprotein을 envelope으로 하는 pseudotyped retrovirus vector system을 이용하여 쥐의 유방상피세포인 HC11에서 human Lactadherin 유전자의 발현을 확인하고자 하였다. 실험에 사용한 vector는 개체내에서의 외래 유전자의 지속적인 발현에 의한 생리적인 부작용을 최소화하기 위한 구조로, 조직특이적이며 lactogenic hormone에 의해 유도적인 활성을 가지는 것으로 알려진 WAP promoter의 통제하에 도입하고자 하는 외래 유전자를 위치하도록 하였다. WAP promoter의 대조군으로 지속적인 활성을 나타내는 β-actin promoter를 사용하였으며, 이 각각의 promoter와 marker gene으로 E. coli LacZ gene을 재조합한 후 retrovirus vector system을 이용하여 HCll에 도입하였다. 세포의 genome 내로의 유전자의 전이는 PCR을 통해 확인하였고, RT-PCR의 수행으로 유전자의 발현을 확인하였다. Lactadherin 유전자를 이용한 실험도 동일한 과정으로 수행하였으며, RT-PCR의 결과에서 HCll 세포에서 Lactadherin 유전자의 발현이 insulin을 단독으로 처리한 군에 비해 insulin, hydrocortisone, prolactin을 동시에 처리한 군에서 우월하게 나타나는 것으로 확인되었다. 그러나 insulin 단독 처리군에서 유전자의 발현이 약하게 나타나는 것으로 관찰되어 WAP promoter의 leakiness에 대한 재고의 필요성이 요구되었다.

4 악 상피세포는 계속해서 탈락하고 세포고사 (Apoptosis) 가 이 과정에 중요한 역 할윤 한다. 본 연구는 콘택프렌즈 착용으로 인한 각막의 부작용에 관여하는 Cytokine 증 어느 Cytokine 이 각막 상피세포의 세포고사를 유도하는지 조사하고자 시행하였다. 각딱 상피세포를 배양하여 4 종류의 인터루킨 OL-1a， IL-1,8, IL-6, IL-H) 과 인터 폐한 감마(l FNr) 그러고 종양괴사 인자 (TNFa‘ TNF，8)를 10 nM, 100 nM, 1μM 농도 포 2. 1, 7 일 동 안 처 리 하였 다. Hoechst :13342 형 광염 색 . Annexin V - FITC/Propi - dium Iodide( Pl) 염 색. TUNEL assay 그퍼 고 DNA Laddering을 시 행 하였 다. IL -la, IL .- 1,8. IL - G. IL - 8는 세 끊파사가 거 의 유도뇌 지 않았고 TNFa, TNF，8는 고농도에 서 약간의 세포고사룹 유도하였으며 IFNr에서는 10nM 에 4 일 동안 노출시 세포고사 가 유도되기 시작하였으며 농도와 노출기간에 비례하여 세포고사 빈도가 높아졌다. IFN -r는 콘택프렌즈로 인한 외상. 감염. 염증이 있을 때 방출되는데 각막상피 세포 고사와 각막병리에 관련이 있는 것으로 사료되며 각막상피세포의 항상성 유지에 중 요한 역할을 하는 것으로 생각된다.

난관상피세포와 그 배양액에서 분비된 고분자 분획이 소정자의 수정능력에 어떠한 영향을 미치는지 알아보고자 실시한 실험에서 다음과 같은 결론을 얻었다. 1. 난관상피세포배양액으로부터 MW 5 kDa cut-off bucket을 사용하여 탈염 및 농축을 실시, 단백질/고분자분획을 회수하고 이를 체외수정용 배양액에 첨가하고 난관상피세포 monolayer와 공배양을 통해 체외수정을 실시한 결과 고분자분획첨가 및 난관상피세포 공배양(OM+OEC)군이 고분자분획첨가