類黃鹼素對A431癌細胞的基質金屬蛋白酶分泌及侵襲性偽足形成能力之影響

Abstract

轉移是癌症致死最主要的一個原因。癌細胞要達成轉移必須經過非常複雜的過程，其中第一步就是癌細胞必須穿越週遭細胞外基質的阻礙。侵襲性偽足(Invadopodia)是位於細胞膜上的微小突起，用於侵入胞外基質；近來有相當多的文獻指出其為主要聚集基質金屬蛋白酶(MMP)並釋放而使細胞可穿越胞外基質的關鍵結構。為了研究轉移的機制，本實驗室將A431子宮頸上皮癌細胞(A431-P)利用Boyden chamber進行篩選，經連續三次穿膜篩選出擁有較高入侵轉移能力之A431第三代細胞(A431-III)。藉由兩株細胞之比較，證明了A431-III具有較高的入侵、移動能力，並且表現較多量的基質金屬蛋白酶；這樣的A431系統提供了一個好的模型以研究癌細胞之轉移入侵過程。在本實驗中，利用A431-P/III的比較發現具強轉移能力的A431-III比起母代細胞形成較多的侵襲性偽足結構以幫助入侵並分解基質。進一步研究這樣的差異主要來自Src蛋白磷酸酶較高的活化程度以及對下游Cortactin蛋白較多的磷酸化。另外也確認了基質金屬蛋白酶(MMP)對於侵襲性偽足的重要性，其中MMP-9是最主要參與的蛋白酶種類。 類黃鹼素(flavonoid)是一群廣泛存在於植物性食物中的天然化合物。根據已發表的報告指出其有相當多的生物活性，其中包括了抗癌及抗癌轉移。本實驗室先前選出兩個具最高抗癌潛能的類黃鹼素:木樨草素(Luteolin)與槲皮素(Quercetin)，對癌細胞活性、生長及轉移具極佳抑制效果，並可抑制基質金屬蛋白酶分泌。鑒於侵襲性偽足對於癌細胞入侵的重要性，本研究證明了此兩種類黃鹼素可以有效抑制侵襲性偽足的產生及抑制細胞外基質的分解。其原因是由於類黃鹼素能抑制包括Src的活化及cortactin的磷酸化，進而抑制侵襲性偽足的形成以及聚集MMP之能力，最終達成抗轉移之結果。

Metastasis is the major cause of mortality in cancer patients and is a complex, multi-step process. Local invasion is the very first step in cancer metastasis. Invadopodia, which are actin-rich membrane protrusions of invasive cancer cells, are responsible for this invasive process. Invadopodia can extend into the extracellular matrix by recruiting matrix metalloproteinases (MMPs) and releasing them; invadopodia are now believed to be crucial structures that allow cancer cells to degrade and penetrate across the extracellular matrix (ECM). We previously obtained a highly invasive A431-III cell sub-line by using the Boyden chamber assay. The A431-III cell line exhibits higher invasive and migratory characteristics. In addition, A431-III exhibits elevated MMP-9 level and epithelial-mesenchymal transition (EMT) phenotype. Thus, this cell line is a reliable model for studying the mechanism of metastasis/invasion. Herein, we showed that A431-III exerted greater ability to form invadopodia leading to more degradation of the ECM than A431 cells. Cortactin, Src, and their phosphorylation have been reported as main regulator of invadopodia formation and function. Our data revealed that the phosphorylation of cortactin (Y421) and Src (Y418) were increased in A431-III cells. Since the degrading ability of invadopodia needs participation of proteases, we also showed the importance of MMPs, especially MMP-9, in this degrading event. Flavonoids, a large group of plant secondary metabolites, are present in almost all-higher plants and display a wide range of pharmacodynamic properties including anti-inflammatory, anti-carcinogenic and anti-metastatic effects. We previously screened out two of the most potent flavonoids, luteolin and quercetin, which can suppress cancer invasion and MMPs secretion. Since it is known that the secretion of MMPs is targeted at invadopodia, our study showed that treating with luteolin and quercetin inhibited the formation of invadopodia in A431-III and decreased the ability of ECM degradation as well. Our data further revealed that these two flavonoids could inhibit Src kinase and the phosphorylation of cortactin, which in turn disrupt the formation of invadopodia. Followed by our previous study that flavonoids inhibited MMPs secretion, these results may explain in part how flavonoids affect MMPs secretion and metastasis potential.