Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/5541
標題: 雌性激素(17b-estradiol)於人類乳癌細胞株誘發基因表現失衡及核酸氧化損害作用之研究
Induction of altered gene expression and oxidative DNA damage by 17b-estradiol in human breast cancer cells
作者: 方茹萍
Fang, Ju-ping
關鍵字: 17b-estradiol
雌性激素
dioxin
oxidative DNA damage
gene expression
戴奧辛
氧化核酸損害
基因表現
出版社: 環境工程學系
摘要: 摘 要 本研究之主旨在於探討環境中之持久性有機污染物(persistent organic pollutants,POPs)-戴奧辛中毒性最大之2.3.7.8-四氯戴奧辛(2.3.7.8-tetrachlorodibenzo-p-dioxin,TCDD),對雌性激素(17β-estrodiol,E2)於人類乳癌細胞株ER(+)/MCF-7 與 ER(-)/MDA-MB-231細胞,誘發基因表現失衡及核酸氧化損害作用之研究。研究結果指出,當MCF-7細胞經TCDD與COMT抑制劑(catechol-o-methyltrasferase inhibitor)預處理後再暴露於E2 (100 M)之下,相對於控制組約可誘發2倍ADL (aldehydic DNA lesions)之生成(p<0.05)。單獨E2 (100 M)之暴露可誘發MDA-MB-231細胞約2倍ADL之產生(p<0.05),但TCDD會抑制由E2於MDA-MB-231細胞所誘發之ADL之生成;當MDA-MB-231細胞於TCDD、COMT抑制劑與E2 (100 M)同時暴露下,相對於控制組,約可誘發2倍ADL之生成(p<0.05)。由E2於MCF-7與MDA-MB-231細胞所誘發之ADL大部分可被putrescine所切除,由上述之結果得知,由TCDD能影響E2代謝及其醌類代謝物所誘發之氧化核酸損害。本實驗亦證實E2 (1-10 nM)確實會造成MDA-MB-231細胞中NAD(P)H之下降(35 %), 然而TCDD可以抑制由E2所造成之NAD(P)H下降。此外,COMT抑制劑之存在則可促進TCDD與E2 (0.1-10 nM)於MDA-MB-231細胞中NAD(P)H之下降(P<0.001),而PRPA-1 〔poly(ADP-ribose)polymerase-1〕抑制劑之存在,可以抑制此一現象。此一結果顯示,TCDD之暴露以及COMT與ER 之存在與否皆會影響E2於生理條件相似之濃度下(1-10 nM),誘發MDA-MB-231細胞之核酸單股斷鏈(DNA single strand breaks,SSBs)。由半定量之RT-PCR結果顯示,TCDD (10 nM)與E2 (100 μM)可以誘發MCF-7細胞中CYP1A1、CYP1B1基因約2.5倍表現量與hOGG1、 hAPE基因約1.5倍表現量;於MDA-MB-231細胞中,單獨之E2可以誘發CYP1A1、CYP1B1基因約1.5倍表現量與hOGG1與 hAPE之mRNA基因分別為4.9與2.1倍之表現量,然而TCDD會抑制由E2於MDA-MB-231細胞中所誘發之hOGG1與 hAPE之mRNA基因表現量。此外,當MDA-MB-231細胞暴露於低濃度(10 nM)且長時間(48 h)之E2環境下,亦可誘發hOGG1與 hAPE之mRNA基因表現(分別為1.74與1.27倍),相同地,TCDD之暴露會抑制由E2於MDA-MB-231細胞中誘發hOGG1與 hAPE之mRNA基因表現量。由RT-PCR之結果得知,TCDD之暴露與ER 之存在與否會影響E2代謝與核酸修補相關基因之表現。綜合以上之實驗結果,對於人類乳癌細胞株而言,TCDD之暴露、ER之存在與否以及COMT之酵素活性,皆是影響雌性激素(E2)被代謝形成醌類代謝物並且誘發細胞核酸氧化損害之重要因素。
Abstract The objective of this research is to investigate the effects of one of the persistent organic pollutants (POPs)- (2.3.7.8-tetrachlorodibenzo-p-dioxin,TCDD), in mediating the induction of imbalances in gene expression and oxidative DNA damage by 17β-estrodiol (E2) in human breast cancer cell lines, including ER(+)/MCF-7 and ER(-)/MDA-MB-231 cells. Results from this study indicated that a 2-fold increase in the number of aldehydic DNA lesions (ADL) was detected in MCF-7 cells exposed to E2 (30 M) pretreated with TCDD and Ro 41-0960, a catechol-o-methyltrasferase (COMT) inhibitor, when compared to the corresponding control (p<0.05). A 2-fold increase in the number of ADL was also detected in MDA-MB-231 cells exposed to E2 (100 μM) alone. In contrast, pretreatment with TCDD (10 nM, 72 h) inhibited the induction of ADL by E2 in MDA-MB-231 cells. In addition, a 2-fold increase in the number of ADL was detected in MDA-MB-231 cells exposed to E2 (30-100 M) pretreated with TCDD and Ro 41-0960 when compared to the corresponding control (p<0.05). Further characterization confirmed that the ADL induced by E2 in MCF-7 and MDA-MB-231 cells was predominantly putrescine-excisable ADL. This evidence indicated that TCDD was capable of modulating the disposition of estrogen to the reactive quinones and the subsequent induction of promutagenic DNA lesions in living cells. In addition, we demonstrated the depletion of NAD(P)H (35 %) in MDA-MB-231 cells exposed to E2 (1-10 nM). Pretreatment with TCDD inhibit the depletion of NAD(P)H in MDA-MB-231 cells exposed to E2. Inclusion of Ro 41-0960 in the incubates enhances the decreases in NAD(P)H levels in MDA-MB-231 cells exposed to E2 (0.1-10 nM) (p<0.001). Further investigation indicates that the E2-induced depletion of NAD(P)H in MDA-MB-231 cells was completely blocked by PARP-1〔poly(ADP-ribose)polymerase-1〕inhibitors. This evidence suggests that exposure to TCDD and the status of COMT and ER modulate the E2-induced DNA single strand breaks (SSBs) in living cells at physiologically revelent concentration (0.1-1 nM). Further, results from semi-quantitative reverse-transcription polymerase chain reaction (RT-PCR) assay indicated that TCDD and E2 induced increases (2.5 folds) in the expression of CYP1A1, CYP1B1, and hOGG1, hAPE genes (1.5 folds) in MCF-7 cells. On the other hand, E2 induced increases (1.5 folds) in the expression of CYP1A1, CYP1B1 and hOGG1, hAPE genes (2.1 and 4.9 folds) in MDA-MB-231 cells. This result suggests that exposure to TCDD and the status of ER alter gene expression in the disposition of estrogen and DNA repair. Overall, our investigation confirmed that exposure to TCDD and the status of COMT and ER modulates the formation of quinonoid derivatives of E2 and the subsequent induction of oxidative DNA lesions in human breast cancer cell lines.
URI: http://hdl.handle.net/11455/5541
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