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

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

中文摘要

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

英文摘要

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.