二十二碳六烯酸對表皮生長因子誘發SK-BR3人類乳腺癌細胞尿激酶型血纖維蛋白溶解酶原活化因子及基質金屬蛋白酶-1/-9表現機制之探討

Effect of docosahexaenoic acid on EGF-induced urokinase plasminogen activator and matrix metalloproteinase-1/-9 expression in SK-BR3 human breast cancer cells

鄭方茹(Fang-Ju Cheng)

指導教授 ： 李健群

中山醫學大學/健康管理學院/營養學研究所/碩士(2013年)

https://doi.org/10.6834/CSMU.2013.00082

摘要

乳癌為全球女性最常見的惡性腫瘤，癌細胞轉移是導致乳癌患者死亡的主要原因。臨床研究發現乳癌病人中約有20~30%具有致癌基因ErbB2的過度表現，其臨床預後差且存活率低。先前研究證實，乳癌細胞發生轉移與否和尿激酶型血纖維蛋白溶解酶原活化因子(Urokinase plasminogen activator, uPA)及基質金屬蛋白酶(Matrix metalloproteinases; MMPs)蛋白質表現與活性的增加有關。另有研究證實EGF可透過活化EGFR/ErbB2，正向調控ErbB2過度表現型SK-BR3人類乳腺癌細胞uPA、MMP-1和MMP-9表現。許多文獻指出，增加二十二碳六烯酸(docosahexaenoic acid, DHA; 22:6n-3)攝取可降低乳癌發生率並改善乳癌預後。雖有不少研究證實DHA具有抑制乳癌細胞轉移能力，但對於抑制SK-BR3乳癌細胞轉移機制仍不清楚。本研究以SK-BR3乳癌細胞為實驗模式，探討DHA對EGF誘發SK-BR3乳癌細胞之EGFR及ErbB2磷酸化與蛋白質表現，及其下游相關訊號路徑所調控uPA、MMP-1及MMP-9酵素活性與蛋白質表現之影響。實驗結果顯示，隨著EGF處理濃度與時間增加，顯著誘發uPA、MMP-1及MMP-9之酵素活性與蛋白質表現，處理DHA (100 μM)可負向調控EGF (40 ng/ml)所誘發uPA、MMP-1及MMP-9之酵素活性與蛋白質表現。DHA可顯著抑制EGF所誘發ERK1/2、JNK、Akt (Ser473)磷酸化，而且細胞預處理EGFR磷酸化抑制劑AG1478也可顯著降低EGF所誘發ERK1/2、JNK、(Ser473)磷酸化。此外，以EGF處理SK-BR3乳癌細胞15分鐘後，即顯著誘發EGFR及ErbB2磷酸化；而EGFR及ErbB2蛋白質表現量亦會隨EGF處理時間增加而增加。一旦加入DHA則可抑制EGF短時間所誘發EGFR及ErbB2磷酸化並降低EGF長時間所誘發EGFR及ErbB2蛋白質表現。免疫螢光染色法與共同免疫沉澱分析法結果顯示，DHA可顯著降低EGF所誘發SK-BR3細胞EGFR與ErbB2蛋白結合並減少胞內分佈。綜合上述結果，DHA經由抑制SK-BR3乳癌細胞之EGFR及ErbB2磷酸化與蛋白質表現與EGFR依賴的ERK、JNK、Akt (Ser473)訊號路徑，進而負向調控EGF所誘發uPA、MMP-1及MMP-9表現，達到抑制SK-BR3乳癌細胞轉移。

關鍵字

二十二碳六烯酸 ； 尿激酶型血纖維蛋白溶解酶原活化因子 ； 基質金屬蛋白酶人類上皮生長因子受體 ； SK-BR3乳癌細胞

並列摘要

Breast cancer is the most commonly diagnosed cancer among women all over the world. Metastasis is the leading cause of death from breast cancer. EGFR and ErbB2 are an important oncogene overexpressed in about 20~30% of breast cancers, and was associated with poor patient outcome and distant metastasis. Previous study showed the induction of urokinase plasminogen activator (uPA) and matrix metalloproteinase (MMP)-1 and MMP-9 activity and expression were associated with breast cancer metastasis. EGF up-regulated uPA, MMP-1, MMP-9 expression by activating EGFR/ErbB2 in ErbB2-overexpressing SK-BR3 breast cancer cells. Diets consisting mostly of docosahexaenoic acid (DHA) have shown to reduce breast cancer incidence rate and have better outcomes There have been several studies on the role of DHA in suppressing breast cancer metastasis,however, the mechanism for the down-regulation of SK-BR3 breast cancer metastasis by DHA is not fully clarified. In the present study, we used ErbB2-overexpression SK-BR3 breast cancer cells to study the effect of DHA on EGF-induced EGFR/ErbB2 expression and activation, and further to investigate whether their downstream signaling pathways are involved in DHA's down-regulation of EGF-induced MMP-1/-9 and uPA expression in SK-BR3 human breast cancer cells. We found that EGF (40 ng/ml) induced MMP-1, MMP-9, and uPA mRNA, protein expression and enzyme activity in both dose- and time-dependent manners, and 100 μM DHA significantly inhibited the induction of EGF. DHA was shown to inhibit EGF-induced activation of ERK1/2, JNK and Akt (Ser473). AG1478, a tyrosine kinase inhibitor of the EGFR, attenuated ERK1/2, JNK and Akt (Ser473) activation as well. The phosphorylation of EGFR and ErbB2 was induced within 15 min after EGF treatment and the total amount of protein was found to increase after 12 h of EGF exposure and these induction were decreased by DHA. Immuno-fluorescence assay and co-immuno-precipitation analysis showed DHA decreases EGFR expression and intracellular localization in the presence of EGF as well as the interaction between EGFR and ErbB2 was disrupted by DHA. These results suggest that attenuation of EGFR and ErbB2 expression and disruption of protein interaction between EGFR and ErbB2 and EGFR-dependent signaling pathways (ERK1/2、JNK、Akt (Ser473)) are involved in DHA's down-regulation of EGF-induced MMP-1/-9 and uPA expression in SK-BR3 human breast cancer cells.

並列關鍵字

Docosahexaenoic acid ； Urokinase plasminogen activator ； Matrix metalloproteinase ； Human epidermal growth factor receptor ； SK-BR3 breast cancer cell

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