血紅素氧化酵素抑制乳癌侵犯之機制

Anti-Tumor Invasion of Heme Oxygenase-1 in Human Breast Cancer

林政緯(Cheng-Wei Lin)

指導教授 ： 侯文琪

共同指導教授 ： 陳彥州

臺北醫學大學/藥學院/藥學研究所/博士(2009年)

摘要

血紅素氧化酵素(Heme oxygenase-1; HO-1)是體內重要的細胞保護與氧化壓力誘導蛋白，其可將血基質(heme)分解成為膽鹽、鐵離子與一氧化碳。過去研究已廣泛證實HO-1具有抗氧化、抗發炎與心血管保護作用等，然而，有關HO-1與腫瘤之間的研究仍具有許多爭議。利用HO-1化學誘導劑(ferric protoporphyrin; FePP)與HO-1大量表現之乳癌細胞(MCF-7/HO-1)抑制腫瘤促進劑TPA所造成之MMP-9表現與細胞侵犯能力，探討其抑制機轉發現HO-1能透過阻斷ROS/ERK/AP-1訊息傳遞路徑進而壓抑MMP-9基因表現，而這些作用都能夠被HO-1抑制劑(SnPP)與HO-1 shRNA處理下有所降低。進一步探討有關HO-1的生化產物中發現，一氧化碳是主要參與在HO-1降低MMP-9表現與腫瘤侵犯能力的主要分子。一氧化碳除了能抑制TPA所誘導的細胞惡性轉移機制外，更能直接影響MMP-9的酵素活性。而比較HO-1在正常乳腺與腫瘤組織也發現HO-1在正常乳腺細胞表現量較高。此外，tumor-associated macrophages (TAM)在腫瘤惡性發展過程中扮演相當重要的角色，TAM所釋放之發炎物質如iNOS與NO也是促進腫瘤侵犯與轉移的因子之一。我們實驗結果發現共同培養乳癌細胞會誘導巨噬細胞活化產生iNOS蛋白與NO表現，而TAM所釋放的NO則能再進一步透過paracrine作用誘導乳癌細胞表現VEGF-A與 MMP-9。處理NOS抑制劑(L-NAME)與HO-1誘導劑(FePP)皆能降低TAM所釋放之iNOS蛋白與NO含量，因而抑制乳癌細胞中VEGF-A與MMP-9的生成。當共同培養乳癌細胞(MDA-MB-231-EGFP)和巨噬細胞也發現會大量促進癌細胞的侵犯性，而處理L-NAME與FePP後也能有效避免其侵犯能力的增加。另外，我們更發現天然物類黃酮素中的quercetin (QUE)能透過誘導HO-1表現、抑制TPA所誘導PKCδ/ERK/AP-1活化，進而降低MMP-9表現與癌細胞移動與侵犯特性。分析QUE的結構相關活性關係更確定3’4’-diOH group是主要負責QUE的生物活性位置所在。從以上三項結果我們推論，誘導HO-1能有效預防乳癌細胞惡性轉型，進一步抑制其侵犯與轉移，因此，開發有效的HO-1誘導劑將能成為未來抗腫瘤轉移之可能策略。

關鍵字

血紅素氧化酵素 ； 乳癌 ； 轉移 ； 基質金屬蛋白脢

並列摘要

Heme oxygenase-1 (HO-1) is an important cytoprotective and stress-responsive enzyme which catalyzes heme into biliverdin, bilirubin, ferric ion, and carbon monoxide (CO). Several beneficial effects of HO-1 such as antioxidant, anti-inflammation, and cardiovascular protection have been identified. The role of HO-1 in tumor progression, however, is still controversial. In this study, effect of HO-1 against the invasion of human breast cancer is investigated. Elevation of HO-1 by the chemical inducer (FePP) or HO-1-overexpressing transfectant (MCF-7/HO-1) significantly suppressed 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced matrix metalloproteinase (MMP)-9 expression and tumor invasion. These events were attenuated by treatment with the HO-1 inhibitor (SnPP) or by transfection of HO-1 shRNA, and blocking the ROS/ERK/AP-1 signaling activation by HO-1 was identified. Furthermore, CO, the byproduct of HO-1, was identified to participate in the suppression of HO-1. CO treatment may suppress MMP-9 enzyme activity in direct and indirect manners. Role of HO-1 in tumor-associated macrophages (TAMs) activation was further investigated. Coculture of breast cancer cells with activated-macrophages stimulated iNOS protein expression and NO production. Release of NO turned to up-regulate VEGF-A and MMP-9 gene expression in breast carcinoma cells, and those were prevented by NOS inhibitor (L-NAME) and HO-1 inducer (FePP). Activated-macrophages promoted the invasive ability of breast cancer MDA-MB-231-EGFP cells, and that was inhibited by the treatment with L-NAME and FePP. Furthermore, a chemical HO-1 inducer quercetin (QUE) is applied to study its anti-invasive effect. QUE has been shown to effectively induce HO-1 protein expression in our previous study. QUE performs significant effect against TPA-induced PKCδ/ERK/AP-1 activation, followed by reducing MMP-9 expression, tumor migration, and invasion. Data of structure-activity relationship (SAR) indicate that 3’4’-diOH groups in QUE play a critical role in suppression of tumor invasion. These data support the notion that HO-1 activation effectively prevents the invasion of breast carcinoma cells, and development of an ideal HO-1 inducer may be beneficial for the treatment of breast cancer invasion.

並列關鍵字

HO-1 ； breast cancer ； metastasis ； MMP

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