論文名稱:

綠茶多酚類對於人類端粒酶促進因子活性之探討

 

The Transcriptional Regulation of the Green Tea Polyphenol on hTERT Promoter Activity

研究生:

洪國富  Kaou-Fu Hung

 

(以作者名查詢陽明大學館藏系統)

 

(以作者名查詢全國圖書書目資訊網)

指導教授:

林姝君  Shu-Chun Lin

        學位類別:

碩士

        學校名稱:

國立陽明大學

系所名稱:

口腔生物研究所

            學號:

39117004

          學年度:

92

          語文別:

中文

          出版年:

93

關鍵字:

綠茶多酚類化合物  Green tea polyphenol

 

人類端粒酶反轉酵素  hTERT

 

  NF-κB

 

  AP-1

全文說明:

(本論文 20090709 對校內公開)

 

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摘要:

茶是普遍性僅次於水的飲料,而綠茶中的多酚類化合物更是被指出具有多種抑制癌症生成的機制,其中包括抑制了與細胞存活息息相關的NF-kBAP-1這兩個轉錄因子;在本實驗室先前的研究發現, 綠茶多酚類中含量最多的(-)-epigallocatechin-3-gallate EGCG)、(-)-epigallocatechin EGC)可以抑制癌細胞hTERT基因的轉錄作用進而導致hTERT mRNA下降,而且在hTERT啟動子上有NF-κBAP-1的結合位置。基於以上的發現,本論文想進一步的探討在口腔癌細胞之中EGCGEGC是否藉由NF-κBAP-1去調控hTERT啟動子.首先探討EGCGEGC對於SASOEC-M1SCC-25OC3這四株口腔癌細胞株的細胞毒性為何,結果發現EGCGEGCSASOEC-M1這兩株分化程度較差的細胞株中的半致死劑量約為30 μM,然而對於SCC-25OC3這兩株分化程度較完全的細胞株其半致死劑量約為160 μM,而進一步利用定量RT-PCR觀察hTERT mRNA102030 μM EGCGEGC處理之下被抑制的情形,結果發現SASOEC-M1在處理20 30 μMEGCGEGChTERT mRNA有顯著的下降;反之,SCC-25OC3hTERT mRNA則都不受影響。除此之外, SAS細胞內的NF-kBAP-1DNA結合活性較OEC-M1強得許多。因此選用SAS進行研究EGCGEGC抑制hTERT基因轉錄的機制。在EMSA實驗結果發現AP-1NF-kB在處理30 μM EGCG6小時其DNA結合能力開始下降,在18小時活性抑制程度最大;而在EGC方面則是在1~2小時活性抑制程度最大,之後活性會慢慢的恢復,進一步則是利用染色質免疫沉澱實驗觀察,這些活性受影響的AP-1NF-κB是否在活體內與hTERT啟動子做專一性結合,結果顯示細胞內的NF-κBAP-1hTERT啟動子無明顯結合。本研究證明了EGCGEGC在較惡性癌細胞中非但可以引起較強的細胞毒性,也會抑制hTERT mRNA的表現。雖然被抑制的AP-1NF-kB活性並不是造成hTERT mRNA 表現下降的主因。

 

Tea is the most widely consumed beverage, second to water. It had been demonstrated that the green tea polyphenols exhibit molecular function of cancer chemoprevention, including suppression of NF-κB and AP-1 which are closely related to cell survival. Our previous investigation has shown (-)-epigallocatechin-3-gallate (EGCG) and (-)-epigallocatechin (EGC), the most common polyphenols in the green tea polyphenols could repress hTERT mRNA expression by inhibiting the transcription of hTERT gene. Since, NF-κB and AP-1 binding motifs are present on the hTERT promoter, this thesis attempts to study further whether EGCG or EGC regulate the hTERT gene via NF-κB and AP-1 signal pathway in the oral cancer cells. Cytotoxicity of EGCG and EGC on various oral cancer cell lines including SASOEC-M1OC3 and SCC-25 was determined. Up to 90% of cells were killed by 80~160 μM EGCG and EGC in SAS and OEC-M1, which are poorly differentiated cell lines. However, in the well- differentiated SCC-25 and OC3 cell lines, only 50% cells were killed even with 160 μM treatment. Real-time quantitative PCR was then used to detect the hTERT mRNA expression in SAS and OEC-M1 cells after treatment with 102030 μM EGCG and EGC. The hTERT mRNA expression in SAS and OEC-M1 cells was inhibited profoundly with 20 or 30 μM EGCG and EGC treatment for 24 h . HoweverEGCG and EGC had no effect on the hTERT gene expression in OC3 and SCC-25 cell lines. SAS cells exhibited higher endogenous DNA binding activity of AP-1 and NF-κB than OEC-M1 cells in EMSA analysis. Therefore, SAS cells, in which the hTERT gene can be down-regulated by EGCG and EGC, were used to investigate the mechanism of hTERT transcription repression. EMSA analysis revealed that the repression of AP-1 and NF-κB binding activity by EGCG and EGC was dose dependent. With 30 μM EGCG, the inhibition appeared at 6h and reached the maximum at 18 h. With 30 μM EGC treatment, the binding activity was inhibited between 1~2 h and recovered gradually. Furthermore chromatin immunoprecipitationChIP was used to observe the specific binding of AP-1 and NF-κB to hTERT promoter in vivo. The result indicated the lack of interaction of AP-1 and NF-κB with hTERT promoter. In conclusion, EGCG and EGC can induce cancer cells death but also inhibit hTERT mRNA expression in the more advanced neoplastic cells. The repression of DNA binding activity of AP-1 and NF-κB did not seem to contribute to hTERT mRNA expression.