木犀草素與槲皮素調控UBE2S表現對高入侵性A431 子代癌細胞轉移及入侵能力之探討

Abstract

癌細胞的轉移是導致人類死亡主要原因之一。當癌細胞開始轉移，他們會分泌基質金屬蛋白(MMPs)及促進皮質-間葉細胞型態轉變(EMT)。在EMT的過程中，會誘導轉錄因子的表現，像是Snail及Twist，而這些與癌細胞的轉移是有關連性的。我們先前的研究顯示攝取木犀草素(Luteolin)與槲皮素(Quercetin)可藉由逆轉皮質-間葉細胞型態轉[epithelial-mesenchymal transision (EMT)]來抑制高入侵能力的癌細胞的轉移。然而木犀草素與槲皮素如何調控癌細胞的轉移仍然是未知的。本研究主要探討癌細胞可經由UBE2S透過EMT來增強轉移能力，而這過程可經由木犀草素與槲皮素來進行抑制。 先前實驗室已取得高侵入性的子宮頸癌上皮皮膚癌細胞A431-III，此細胞能大量表現MMP-9及比母代有較佳轉移能。我們發現較高入侵能力的A431第三代細胞(A431-III)會比A431母代細胞(A431-P)明顯較高的UBE2S表現量。經由UBE2S siRNA阻斷UBE2S的表現，及大量表現UBE2S實驗中，顯示UBE2S能藉由EMT來增加細胞的入侵及移動能力。處理木犀草素與槲皮素後可以明顯抑制UBE2S的表現量。抑制A431-III的UBE2S表現及A431-P大量表現UBE2S的實驗中，也顯示了能負調控VHL的表現，而與Hif-1α的表現呈正相關。 UBE2S泛素載體蛋白是為E2泛素連接酶,可經由連結蛋白質後，進入蛋白酶體進行降解，近年來被報導與癌細胞的形成及進展有相關性。而在此篇研究中，我們發現在惡性腫瘤中大量表現UBE2S藉由誘導EMT的發生促進癌細胞的移動及入侵能力。同時，我們數據亦證實，UBE2S能促進IκB的降解及活化NF-κB的路徑，進而影響Snail的表現及促進EMT的發生。 木犀草素與槲皮素可以抑制這反應。在子宮頸癌細胞中顯示UBE2S與VHL及Hif-1α的表現有相關性。這些結果顯示UBE2S能幫助子宮頸癌細胞的活動能力，並可經由木犀草素與槲皮素來抑制子宮頸癌細胞的轉移。我們的實驗結果進一步證實UBE2S可作為腫瘤細胞轉移治療的標的。

Cancer metastasis is the main cause of human death. When cancer cells start to migrate, they could secret matrix metalloproteinases (MMPs) and promote EMT process. Expression of the transcription factors such as Snail and Twist was enhanced during EMT, and this correlates with cancer metastasis. We have previously reported that the dietary flavonoids luteolin and quercetin might inhibit the invasiveness of highly invasive cervical cancer by reversing EMT signaling. However, the regulatory mechanism of luteolin and quercetin on cell invasiveness is still unclear. The present study demonstrates that luteolin and quercetin inhibits EMT signaling and thereby cancer cell invasion through UBE2S. We previously prepared a highly invasive A431-III cell line, which expression a high level of MMP-9 and exhibit high migration ability than the parent line A431-P. Here, we found that UBE2S expression was significantly higher in highly invasive A431-III cells than A431-P cells. The UBE2S siRNA knockdown and overexpression experiments show that UBE2S increased the migration and invasion ability of cancer cells through EMT signaling. Dietary flavonoids luteolin and quercetin could significantly inhibit UBE2S expression. Knockdown and overexpression of UBE2S in A431-III and A431-P cells show negative correlation with VHL expression and positive correlation of Hif-1α expression. The UBE2S ubiquitin carrier protein is an E2 ubiquitination ligase that helps the E1, E2 and E3 ligases link ubiquitin with target proteins, which then targets protein towards proteasome degradation, is implicated in cancer cell formation and progression. Our findings suggest that high UBE2S expression in malignant cancers contributes to cell motility and invasion ability through EMT signaling. The findings are also suggestive that UBE2S triggers the degradation of IκB and activation of NF-κB pathway, resulting in the subsequent induction of Snail expression and promotion of EMT in A431-III cells. Luteolin and quercetin could offset this effect. UBE2S show correlation with expression of VHL and Hif-1α in cervical cancer cells. These results show the metastatic inhibition of cervical cancer by the flavonoids luteolin and quercetin and provide a functional role of UBE2S in cell motility of cervical cancer. These results support UBE2S could be a potential therapeutic target in cervical cancer.