Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/5077
標題: 代謝活化及基因表現變異對多氯聯苯誘發人類乳腺癌細胞氧化壓力、DNA損害及細胞毒性作用之影響
Effects of metabolic activation and altered gene expression on the induction of oxidative stress, DNA damage, and cell toxicity in human breast cancer cells by polychlorinated biphenyls (PCBs)
作者: 黃景隆
Huang, Ching-Lung
關鍵字: human breast cancer cells
人類乳腺癌細胞
MCF-7 cells
T47D cells
MDA-MB-231 cells
PCB52
PCB77
oxidative stress
DNA damage
apoptosis
ER
多氯聯苯
氧化壓力
DNA損害
細胞凋亡
雌性激素受體
出版社: 環境工程學系
摘要: 本研究之主旨在於探討低氯數之多氯聯苯同屬物(Polychlorinated biphenyls, PCBs)對人類乳腺癌細胞, 包括ERα(++)/MCF-7, ERα(+)/T47D 及 ERα(-)/MDA-MB-231,經由代謝活化過程及改變特定基因表現,能否進而誘發細胞DNA損害及細胞毒性之機制。細胞毒性測試結果顯示,三株人類乳腺癌細胞(MCF-7, T47D, MDA-MB-231)與2,2,5,5-tetrachlorinated biphenyl (PCB52)或3,3,4,4-tetrachlorinated biphenyl (PCB77)反應,皆產生顯著之細胞毒性,且細胞毒性效應隨反應時間與添加之劑量成正相關(time- and concentration-dependent effects),且PCB52所產生之細胞毒性明顯大於PCB77,且細胞凋亡(apoptosis)為導致細胞毒性的主要途徑。若T47D 及 MDA-MB-231細胞進一步以CYP1A(α-Naphthoflavone, resveratrol)及CYP2B抑制劑(metyrapone)預處理後,可保護細胞免於受PCB52及PCB77所誘發之細胞毒性,但於MCF-7細胞中,CYP1A/2B抑制劑則無法抑制由PCB52所誘發之細胞毒性,且此一結果可能與ERα存在與否有關。除此, PCB52及PCB77與MCF-7,T47D及MDA-MB-231 細胞反應後,皆可誘發細胞內ROS (reactive oxygen species)量增加,且以MDA-MB-231細胞內產生之ROS量最多,而細胞以CYP1A/2B抑制劑預處理後皆可分別減少PCB77及PCB52所誘發之ROS量,顯示ROS之累積為細胞毒性主要來源之一。另外,以無細胞毒性之PCBs濃度與細胞反應,觀察細胞內NAD(P)H消耗情形,經由PARP-1活化分析結果顯示,PCB52 (1 μM)及PCB77 (10 μM)及兩者混合皆不能誘發MCF-7 細胞之NAD(P)H下降。而在T47D及MDA-MB-231細胞中,單獨PCB52及PCB77皆可誘發NAD(P)H及 NAD+下降,且三種PARP抑制劑皆能有效抑制此一效應,但於PCB52及 PCB77混合條件下,並無明顯之NAD(P)H及NAD+下降,此一結果顯示,單獨PCB52及PCB77皆可誘發T47D及MDA-MB-231細胞DNA股斷鏈,導致PARP-1活化,但於PCB52及 PCB77混合條件下,具相互拮抗效應。進一步由半定量之反轉錄-聚合酶連鎖反應(semi-quantitative RT-PCR)結果顯示,於MCF-7細胞中,PCB77(10 μM)可誘發CYP1A1、hAPE及XRCC1 mRNA之基因表現量增加;而PCB52(1 μM)可誘發hOGG1、hAPE及XRCC1 mRNA之基因表現量增加;而混合效應可誘發hOGG1及XRCC1 mRNA之表現量增加。MDA-MB-231細胞中,PCB77(10 μM)及PCB52(10 μM)皆可誘發CYP1A1及XRCC1 mRNA之表現量增加;而混合效應可增加CYP1A1 mRNA之表現量。由以上結果顯示,於人類乳腺癌MCF-7細胞中,PCB52及PCB77皆能明顯誘發基因表現失衡,而導致細胞內ROS之產生,並直接引發細胞凋亡;相反地,人類乳腺癌MDA-MB-231細胞中,ROS之主要來源可能為PCB52或PCB77於代謝活化過程,經由氧化還原循環(redox cycling)及無效循環(futile cycling)所產生,進一步誘發DNA股斷鏈,而最終導向細胞凋亡。除此,本實驗結果也顯示,ERα於調控多氯聯苯所誘發的DNA損害及細胞毒性上扮演著重要角色。
The objective of this research is to examine the effects of metabolic activation and altered gene expression on the induction of oxidative stress, DNA damage, and cell toxicity by polychlorinated biphenyls (PCBs), including PCB52 and PCB77, in human ERα(++)/MCF-7, ERα(+)/T47D and ERα(-)/MDA-MB-231 breast cancer cells. Results indicated that both PCB52 and PCB77 induced concentration- and time-dependent increases in cytotoxic response in MCF-7, T47D, and MDA-MB-231 cells. The extent of cytotoxic response induced by these two PCB congeners in human breast cancer cells was greater for PCB52 than for PCB77. Additionally, the reduction in the number of viable cells exposed to PCBs was primarily mediated by apoptosis. Further, α-naphthoflavone, resveratrol, and metyrapone blocked the PCB52-induced cell toxicity in T47D and MDA-MB-231 cells, but not in MCF-7 cells. The data also showed that PCB52 and PCB77 induced significant increases in intracellular levels of reactive oxygen species (ROS) in MCF-7, T47D, and MDA-MB-231 cells, particularly in MDA-MB-231 cells. In addition, when cells were exposed to PCBs at non-cytotoxic concentration, we observed that PCB52 (1 mM) and PCB77 (10 mM) alone or mixture did not induce decreases in intracellular NAD(P)H in MCF-7 cells. In contrast, both PCB52 and PCB77 induced decreases in intracellular NAD(P)H and NAD+ through formation of DNA strand breaks and poly(ADP-ribose)polymerase-1 activation in T47D and MDA-MB-231 cells. Antagonism was detected in T47D and MDA-MB-231 cells exposed to PCB52/PCB77 mixture. Further, results from semi-quantitative reverse-transcription polymerase chain reaction (semi-quantitative RT-PCR) analyses indicated that exposure to PCB77 (10 mM) induced increases in the expression of CYP1A1, hAPE, and XRCC1 whereas PCB52 (1 mM) alone induced increases in the expression of hOGG1, hAPE, and XRCC1. Similar observation was also detected in MCF-7 cells exposed to PCB52/PCB77 mixture. On the other hand, both PCB52 (10 mM) and PCB77 (10 mM) induced increases in the expression of CYP1A1 and XRCC1 genes whereas PCB52/PCB77 mixture induced increases in the expression of CYP1A1 in MDA-MB-231 cells. Overall, this evidence indicates that both PCB52 and PCB77 may induce ROS formation and imbalances in the expression of genes responsible for the DNA repair process and apoptosis in MCF-7 cells. In contrast, PCB52 and PCB77 are likely to mediate the induction of ROS formation, DNA strand breaks, and cell toxicity through futile cycling and/or redox cycling in MDA-MB-231 cells. The data also suggests that the status of estrogen receptor a may play a role in modulating the PCB-induced DNA damage and cytotoxic response in human breast cancer cells.
URI: http://hdl.handle.net/11455/5077
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