類黃鹼素對A431癌細胞的活性氧影響及轉移和入侵能力之探討

Effects of flavonoids on reactive oxygen species and metastasis-associated ability in highly invasive A431-III sub-line

鄭維鈞(Wei-Jun Zhang)

指導教授 ： 李明亭

國立臺灣大學/生命科學院/生化科學研究所/碩士(2015年)

https://doi.org/10.6342/NTU.2015.11316

摘要

癌細胞的轉移一直是癌症致死的主要原因，因此了解癌細胞的轉移機制一直是個重要的研究課題。在過去的文獻中指出，癌細胞內的活性氧 (reactive oxygen species，ROS) 程度和其惡化有密切的關係，在較惡性的癌細胞內常偵測到較高程度的活性氧，在癌細胞惡化的過程中，活性氧含量會改變並活化不同的訊息傳遞途徑，且提升基質金屬蛋白酶 (matrix metalloproteinase，MMP) 的表現及釋放，進而造成癌細胞的入侵和轉移能力上升。
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，經過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)。A431-III具有較高的入侵和移動能力，並表現較高量的基質金屬蛋白酶，這樣的A431細胞系統提供了一個好的研究模型來探討癌細胞之轉移入侵過程。藉由兩株細胞之比較，我們發現A431-III相較於A431-P產生較多的活性氧，實驗結果發現A431-III的抗氧化酵素MnSOD表現量上升，而catalase表現量下降，因此A431-III細胞內活性氧量上升可能是因為抗氧化酵素表現量改變所造成的，這些抗氧化酵素是細胞平衡內部活性氧含量的一道重要清除機制。
類黃鹼素 (flavonoid) 是一群廣泛存在於蔬果中的天然化合物，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轉移。本實驗室先前篩選出兩個具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、生長及轉移均具極佳抑制效果。初步研究發現Luteolin和Quercetin能有效降低A431-III內部的活性氧含量，此外Luteolin和Quercetin處理也增加了A431-III的catalase的表現量，其結果表示了Luteolin和Quercetin能藉由改變細胞內抗氧化酵素的表現量來影響活性氧的清除，之後我們使用H2O2處理A431-P細胞提高細胞內部的活性氧含量，使用NAC、DPI處理A431-III細胞降低細胞內部的活性氧含量，結果A431-P細胞的移動能力上升，而A431-III細胞的移動能力下降，表示類黃鹼素清除活性氧的能力和其抑制癌細胞生長和轉移的抗癌活性有所關聯。

關鍵字

類黃鹼素 ； 腫瘤 ； 癌症 ； 活性氧

並列摘要

Tumor metastasis has always been the main factor that causes the death of cancer patients, thus it is important to realize the mechanism of cancer metastasis. It has been appreciated for a number of years that reactive oxygen species (ROS) production is increased in malignant cancer cells. During tumor progression, reactive oxygen species can activate MMP activity, cell-ECM adhesion, and subsequently promotes the capability of cell migration and cell invasion. The signaling pathway involved in these processes are thought to be achieved through redox modification of signaling molecules such as protein kinases and transcription factors.
In the previous study from our laboratory, we have selected highly invasive tumor cell sub-line A43-1III from A431-P by using Boyden Chamber Assay (unpublished data). In this study, we take the advantage of this highly invasive sub-line to further explore the role of reactive oxygen species in cancer cells. We find that reactive oxygen species production is increased in A431-III sub-line compared to A431-P. This result may attribute to differential expression of antioxidants, which were important in balancing cellular ROS levels. We find that MnSOD expression is increased whereas catalase is decreased in A431III sub-line compared to A431P.
The flavonoids are polyphenolic compounds that are ubiquitous constituents of flowering plants, particularly food plants. Plant flavonoids have been recognized as possessing antitumor effects. Two dietary flavonoid constituents, luteolin (Lu) and quercetin (Qu), generally appear to be the most potent among plant flavonoids in terms of their in vitro biological activities. ROS level was decreased in A431-III sub-line after treatment of luteolin or quercetin. Furthermore, luteolin or quercetin treatment in A431-IIII sub-line promoted catalase expression on protein level. These results suggest that luteolin and quercetin can influence ROS elimination in cells by regulating the expression of antioxidant enzymes. Then, we tested the effect of ROS level in A431 cells. We promoted ROS level in A431-P by direct H2O2 treatment, and suppressed ROS level in A431-III by treating antioxidant, NAC and DPI. The capability of migration was promoted in A431-P and suppressed in A431-III. Experimental results are shown that the ability of flavonoids scavenging reactive oxygen species is associated with their anti-tumor activity.

並列關鍵字

flavonoid ； tumor ； cancer ； reactive oxygen species

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