하인두암에서 예후인자로서의 표피성장인자수용체(EGFR) 과발현과 하인두암의 진행에 있어 표피성장인자(EGF)의 역할 분석 Overexpression of EFGR as Prognostic Factor and Effect of EGF in the Progression of Hypopharyngeal Cancer원문보기
연구배경 및 목적 표피성장인자수용체(EGFR)는 HER2/neu(erbB2), HER3(erbB3), HER4(erbB4)를 포함하는 receptor tyrosine ki-nase의 erbB 그룹에 속하는 수용체이다. 표피성장인자수용체의 과발현은 다양한 종류의 암, 특히 두경부편평세포암에서 예후를 악화시킨다고 알려져 있다. 이에 저자들은 하인두편평세포암에서 표피성장인자수용체의 발현 및 분포를 확인하고, 하인두암에서 표피성장인자(EGF)가 암세포의 증식과 침습에 미치는 영향에 대해 알아보고자 하였다. 대상 및 방법 57명의 하인두편평세포암 환자의 조직에서 표피성장인자수용체의 발현을 면역화학적염색을 통해 확인하고, 이에 대해 임상병리학적 요인과 생존율에 대한 분석을 시행하고, 일부 환자의 정상 및 암조직에서 Western blot을 시행하였다. 하인두편평세포암 세포주인 FaDu에서 proliferative assay, colony dispersion, wound healing assay, invasion assay를 시행하여, 하인두암의 진행에서 표피성장인자의 역할에 대하여 분석하였다. 또한 RT-PCR과 Zymography를 통하여 Matrix metalloproteinase(MMP)-2, 9의 발현을 확인하였다. 결 과 63.2%의 하인두편평세포암 조직에서 표피성장인자수용체의 발현이 확인되었다. 표피성장인자수용체의 발현은 정상조직에서 비하여 하인두암 조직에서 유의하게 증가되어 있었으며, 병리학적 병기(p=0.022)가 올라갈수록 유의하게 증가하였으나, 증례수의 제한으로 생존율에서는 통계적 유의성을 얻지는 못했다(p=0.053). in vitro의 결과로 표피성장인자를 FaDu 세포주에 처리하였을 때, FaDu 세포주의 증식이 유의하게 증가되었으며(p<0.05), Transwell invasion chamber상 침습의 증가가 확인되었다(p<0.05). RT-PCR과 zymogram 실험상 표피성장인자처리시 FaDu 세포주의 MMP-2, 9이 발현이 증가되고 활성화되는 것이 확인하였다. 결 론 본 연구에서 표피성장인자수용체의 과발현이 하인두암의 예후 인자로서의 가능성을 확인하였고, 표피성장인자가 하인두편평세포암의 증식과 침습에 관여하는 것을 확인하였다.
연구배경 및 목적 표피성장인자수용체(EGFR)는 HER2/neu(erbB2), HER3(erbB3), HER4(erbB4)를 포함하는 receptor tyrosine ki-nase의 erbB 그룹에 속하는 수용체이다. 표피성장인자수용체의 과발현은 다양한 종류의 암, 특히 두경부편평세포암에서 예후를 악화시킨다고 알려져 있다. 이에 저자들은 하인두편평세포암에서 표피성장인자수용체의 발현 및 분포를 확인하고, 하인두암에서 표피성장인자(EGF)가 암세포의 증식과 침습에 미치는 영향에 대해 알아보고자 하였다. 대상 및 방법 57명의 하인두편평세포암 환자의 조직에서 표피성장인자수용체의 발현을 면역화학적염색을 통해 확인하고, 이에 대해 임상병리학적 요인과 생존율에 대한 분석을 시행하고, 일부 환자의 정상 및 암조직에서 Western blot을 시행하였다. 하인두편평세포암 세포주인 FaDu에서 proliferative assay, colony dispersion, wound healing assay, invasion assay를 시행하여, 하인두암의 진행에서 표피성장인자의 역할에 대하여 분석하였다. 또한 RT-PCR과 Zymography를 통하여 Matrix metalloproteinase(MMP)-2, 9의 발현을 확인하였다. 결 과 63.2%의 하인두편평세포암 조직에서 표피성장인자수용체의 발현이 확인되었다. 표피성장인자수용체의 발현은 정상조직에서 비하여 하인두암 조직에서 유의하게 증가되어 있었으며, 병리학적 병기(p=0.022)가 올라갈수록 유의하게 증가하였으나, 증례수의 제한으로 생존율에서는 통계적 유의성을 얻지는 못했다(p=0.053). in vitro의 결과로 표피성장인자를 FaDu 세포주에 처리하였을 때, FaDu 세포주의 증식이 유의하게 증가되었으며(p<0.05), Transwell invasion chamber상 침습의 증가가 확인되었다(p<0.05). RT-PCR과 zymogram 실험상 표피성장인자처리시 FaDu 세포주의 MMP-2, 9이 발현이 증가되고 활성화되는 것이 확인하였다. 결 론 본 연구에서 표피성장인자수용체의 과발현이 하인두암의 예후 인자로서의 가능성을 확인하였고, 표피성장인자가 하인두편평세포암의 증식과 침습에 관여하는 것을 확인하였다.
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제안 방법
In this study, even in the cases when EGFR expression was not observed, EGFR was presumed to be present, but could not be observed because it was expressed at levels that were undetectable by immunohistochemistry. Further studies measuring the concentration of EGFR in the tissue or using Western blotting to determine the relationship between the concentration of EGF and EGFR, and their expression are warranted. Although no statistical significance was detected between the expression of EGFR and the survival rate in this study(#=0.
In this study, immunohistochemical staining was performed on 57 cases of hypopharynx cancer to analyze the expression of EGFR. Overexpression of EGFR was noted in 63.
To assess the contributions of EGF to both migratory and proliferative activities, we performed an in wound healing assay in FaDu cells using EGF at 0, 10, and 30ng/mL concentrations. Exogenous EGF significantly enhanced the migration and proliferation of FaDu cells in dose-dependent manner(Fig.
The monolayer was scratched with a pipe卄e tip and washed with PBS. To evaluate the effect of EGF on both migratory and proliferative activities, FaDu cells were treated with EGF at 0, 10, and 30ng/mL concentrations. Exogenous EGF enhanced the migration and proliferation of FaDu cells in a dose-dependent manner(p<0.
대상 데이터
FaDu cells, a hypopharyngeal cancer cell line, were obtained from the American lype Culture Collection(ATCC, Manassas, VA, USA) and cultured in Eagle's minimum essential medium (EMEM) containing 25mM NaHCO3, 10% fetal bovine serum (FBS), glutamine, 1% essential amino acids, vitamin E, and streptomycin at 37℃ under a 5% CO2 atmosphere.
Fifty-seven patients admitted for hypopharyngeal squamous cell carcinoma between 1994 and 2002 at Ajou University Hospital and Yonsei University Medical Center in Korea were enrolled in this study. Surgically removed specimens of hypopharyngeal squamous cell carcinoma, excluding carcinoma in situ and verrucous carcinoma, were used.
1%) died during the follow-up period. The cause of death was local recurrence in 11 cases and distant metastasis in nine cases. Survival analysis was performed using the Kaplan-Meier method, and the statistical significance was evaluated by the log-rank test(Fig.
데이터처리
The cause of death was local recurrence in 11 cases and distant metastasis in nine cases. Survival analysis was performed using the Kaplan-Meier method, and the statistical significance was evaluated by the log-rank test(Fig. IB). Although a difference in survival rates appeared to exist between the groups with negative and positive EGFR expression, the_p value was 0.
이론/모형
Patient survival rates were calculated using the Kaplan-Meier method, and statistically significant differences in survival were identified using the log-rank test. Studenfs /-test and one-way analysis of variance(ANOVA ; for the invasion assay) were also used for analyses.
Pearson's chi-square test and Fisher's exact test were used for statistical analyses of immunohistochemical data. Patient survival rates were calculated using the Kaplan-Meier method, and statistically significant differences in survival were identified using the log-rank test.
The reaction mixture was centrifuged at 12,000 rpm for 5 min at 4℃, and the supernatant was used for Western blot analysis. Protein concentration was measured using the Bio-Rad protein assay. Sodium dodecyl sulfate(SDS)-polyacrylamide gel electrophoresis (PAGE) was used to separate 20 eg of protein per well, and the bands were transferred to a nitrocellulose filter(Amersham, Piscataway, NJ, USA).
None of the patients had undergone radiotherapy or chemotherapy before surgical excision. The tumors were classified according to the 2002 AJCC TNM classification system. The average age at clinical onset was 60 years, with a range of 37—76 years.
성능/효과
Because EGFR was more strongly expressed in the cancer cells, EGFR activation is thought to be associated with the progression of cancer. In this study, even in the cases when EGFR expression was not observed, EGFR was presumed to be present, but could not be observed because it was expressed at levels that were undetectable by immunohistochemistry. Further studies measuring the concentration of EGFR in the tissue or using Western blotting to determine the relationship between the concentration of EGF and EGFR, and their expression are warranted.
Pathologic cell differentiation did not seem to affect EGFR expression. No statistically significant difference was observed between groups with and without recur- rence(60% and 50%, respectively). Of the 57 patients, 20(35.
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