七層塔水萃物減少人類肺腺癌A549細胞株MMP-2與u-PA蛋白表現之訊息路徑探討

Investigation of MMP-2 and u-PA expressions inhibited by Aqueous O. gratissimum extract in human lung carcinoma A549 cells

柯美慧(Mei-Hui ko)

指導教授 ： 高紹軒

中山醫學大學/醫學院/生化暨生物科技研究所/碩士(2010年)

https://doi.org/10.6834/CSMU.2010.00103

摘要

肺癌在台灣所有癌症死亡患者中排名第一，而肺腺癌又佔所有肺癌類型約40 %。癌細胞高度的侵襲及轉移能力，是導致癌症病患預後不良或是致死的主因之一；因此，如何有效降低癌細胞的侵襲及轉移能力，對於治療癌症以及降低其復發率是很重要的。羅勒屬植物的七層塔已有多篇研究文獻指出它的萃取物具有抗癌的生物活性，本實驗室先前的研究也證實七層塔水萃物可促使人類肺腺癌A549細胞進行凋亡作用，進而抑制癌細胞的生長，而本研究則進一步研究七層塔水萃物是否具有抑制A549細胞轉移的能力及其作用機轉。在較低的七層塔水萃物作用後，以MTT assays發現對A549的細胞存活率無顯著影響。而wound healing assay的結果顯示低濃度七層塔水萃物便可以有效降低A549細胞的移動能力。透過zymography assay發現低濃度七層塔水萃物可以減少A549細胞分泌第二型基質金屬蛋白酶(MMP-2)與尿激素型胞漿素原活化蛋白(u-PA)。另外，我們也利用西方墨點法探討低濃度七層塔水萃物對A549相關的訊息傳遞路徑之影響，結果發現AKT蛋白磷酸化明顯減少且ERK蛋白磷酸化有上升變化。因此，本研究結果顯示低濃度七層塔水萃物不會影響A549細胞的存活率，但是會減少第二型基質金屬蛋白酶及尿激素型胞漿素原活化蛋白的活性，推測因此導致A549細胞移動能力的下降。然而對相關訊息傳遞路徑的影響與先前的研究結果不完全一致，有待進一步的深入探討與確認。而本研究結果也初步顯示低濃度七層塔水萃物或可應用在降低肺癌轉移的相關輔助治療。

關鍵字

七層塔 ； 第二型基質金屬蛋白酶

並列摘要

Lung cancer is the leading cause of death in Taiwan. Among the all types of lung cancers, adenocarcinoma is the most common (~40%) and lethal type. Metastasis is a major capability of cancer cells that causes poor prognosis, recurrence and even death. Therefore, effective inhibition of metastasis should be very helpful and useful in cancer therapy. Ocimum gratissimum Linn (OG), having been widely used in herbal medicine, has been demonstrated to possess multiple anticancer bioactivity. Our previous studies have shown that aqueous extract of OG (OGE) is able to induce apoptosis of human lung adenocarcinoma A549. In the present study, we further investigate whether OGE inhibits metastasis of A549 cells and the underlying mechanism. Cell viability assay by MTT revealed that low-dose of OGE (less than 200 µg/ml) insignificantly affected viability of A549 cells. Interestingly, the low-dose OGE treatments significantly inhibited the cell motility of A549 cells by wound healing assay, as well as reduced the activity of secreted matrix metalloproteinases (MMPs) and urokinase-plasminogen activator (u-PA) by zymography assay. Additionally, we also investigated signaling pathways associated with expression of MMPs and u-PA, which showed that the low-dose OGE treatments inhibited phosphorylation of AKT but enhanced that of ERK. In conclusion, our findings indicate that low-dose OGE significantly attenuates mobility of lung carcinoma cell A549, which may attribute to inhibition of secreted MMP-2 and u-PA by low-dose OGE. It is suggested that low-dose OGE is beneficial to a combination treatment against lung cancer metastasis.

並列關鍵字

O. gratissimum ； MMP-2

參考文獻

2.郭壽雄：肺癌。當代醫學1998；13：8。

26.伯穎、謝文斌：外科與非外科治療肺癌存活情形。當代醫學雜誌2000；335：717。

1.Hongchang Shen, Dongyan Hu, Jiajun Du, Xiuwen Wang, Yan-guoLiu,Yawei Wang, Jun-min Wei, Daoxin Ma, Pu Wang, Lei Li. (2008) , Paclitaxel-octreotide conjugates in tumor growth inhibition of A549 human non-small cell lung cancer xenografted into nude mice.European Journal of Pharmacology . 601 (1-3) : 23-9.

4.Anthelmintic activity of essential oil of Ocimum gratissimum Linn. and eugenol against Haemonchus contortus. Veterinary Parasitology. 109 (1-2) : 59-63.

6.Miriam R. Bueno, Adrian Daneri and Juan Armendáriz-Borunda.(2000), Cholestasis-induced fibrosis is reduced by interferon α-2a and is associated with elevated liver metalloprotease activity. J.Hepatol. 33 (6) : 915-925.