松香酸抑制黑色素瘤B16F10細胞侵入和移動能力及其機制探討

Study of the inhibitory effect and its mechanism of Abietic acid on B16F10 melanoma cancer cell invasion and migration

楊昇翰(Sheng-Han Yang)

指導教授 ： 謝易修 ； 陳霈霓

中山醫學大學/醫學院/生化暨生物科技研究所/碩士(2010年)

摘要

黑色素癌 (melanoma)為一惡性腫瘤源自於黑色素細胞，此類細胞會產生黑色素於人體之皮膚、眼睛以及毛髮。黑色素癌是可被預防的、避免接觸過量的紫外線或日光浴為主要方法。惡性的黑色素癌具有侵襲性並會擴散至全身淋巴結，較其他皮膚癌更常出現轉移的情況。全球每年大約有一萬六千名惡性黑色素瘤的新個案被診斷出，而又以白種人男性發生率居高，而根據WHO的統計、全球每年約有四萬八千人死於黑色素癌，而目前主要的治療方法包含手術切除、化學療法、免疫療法與放射線治療。 癌細胞的轉移為惡性腫瘤的重要特性之一，因此、在很多研究中大都針對癌細胞的轉移過程，開發新藥或是藥性溫和的天然藥物處理，對人體無傷害性，可以增強人體抵抗力的藥物活性成分。松香酸是一種天然的樹脂酸，而松香主要是由松樹木材和樹脂中分離出來，他是由許多結構相近的有機酸所構成，先前在許多文獻上證實松香酸具有許多生物活性，最常見的有抗菌、抗過敏、抗發炎、抗腫瘤等功效，但松香酸抑制癌細胞侵襲與轉移的機制至今還不是很清楚。本實驗以極度惡性的老鼠黑色素癌細胞 (B16F10) 做研究，利用松香酸來看其侵襲與轉移的能力以及機制作一系列研究。首先以B16F10做Wound healing assay，發現松香酸抑制細胞移動效果顯著，在MTT assay中不影響細胞存活率。接著利用Boyden chamber assay觀察到在處理松香酸20μM時invasion 和 migration 被抑制 (P<0.05) 。用Zymography assay 看MMP-9與u-PA 活性，細胞處理松香酸30μM時MMP-9就有明顯下降，而u-PA 在處理松香酸10μg/ml時有意義 (P<0.05) 。以Western blot看其路徑，主要透抑制p-Src、PI3K、pAkt蛋白表現量進而抑制核內轉錄因子AP-1的結合能力。另外在C57BL/6J 老鼠中，以尾靜脈注射，隨機分組，肺部組織切片有因為餵藥而使黑色素瘤轉移的部分明顯減少，20mg 組也回到正常切片的組織狀況。在活體實驗中，松香酸明顯的抑制腫瘤生長，及侵襲與轉移能力明顯受到抑制。在實驗中再以人類黑色素癌細胞(A2058)處理松香酸，以MTT assay、Zymography assay、boyden chamber assay實驗同樣發現相似結果，可以推測松香酸對黑色素癌細胞都有抑制轉移與侵襲的效果。在未來可以發現更多松香酸的其他治療功能，也能對其他的疾病有很好的療效，使其在研究上可以對人類有更多的福祉貢獻。

關鍵字

松香酸 ； 黑色素瘤 ； 尾靜脈

並列摘要

Melanoma, the most serious type of skin cancer which produce the pigment melanin that colors our skin, hair, and eyes. It is often preventable by protecting children and adults from sunburn and excessive sun exposure. It has spread to nearby lymph nodes that help fight infection. Around 160,000 new cases of melanoma are diagnosed worldwide each year, and it is more frequent in males and Caucasians. According to a WHO report about 48,000 melanoma related deaths occur worldwide per year. The treatment includes surgical removal of the tumor, adjuvant treatment, chemo- and immunotherapy, or radiation therapy. The metastasis of cancer is a vital trait in malignance.Therefore，the development of new remedies or and folk medicine for a long time.The dose not have the injury to the human body. Abietic acid,a group of natural resin acid and is most abundant of several closely-related organic acid that constitute most of rosin.Previously,many studies has been evidented to possess various prominent bio-activities.Antimicrobial,antiallergic,anti-inflammatory and antitumor activities are the most representative for it.But,the in vivo effect and detailed mechanism of Abietic acid on metastasis and invasion was still unclear. In this study, we determined the effect of Abietic acid on cell migration; meanwhile, showed that Abietic acid could efficaciously inhibit melanoma cell migration and invasion by wound healing assay and Boyden chamber invasion assay. From this result, Abietic acid were chosen for the subsequent experiments. whereas the effect of Abietic acid on migration and invasion of tumor cells has not been clearly clarified. This study first demonstrates that, in the absence of cytotoxicity, Abietic acid exerted a dose-dependent inhibitory effect on the invasion, migration, and motility of highly metastatic murin melanoma cells (B16F10) and human melanoma cells (A2058). We examined the effect of Abietic acid on factors of cancer metastasis. We treated tumor cells with various concentrations of Abietic acid, for set periods, and then subjected cells to gelatin zymography, casein zymography, and Western blot to investigate the expression of matrix metalloproteinase-2 (MMP-2), urokinase-type plasminogen activator (u-PA). Following treatment with Abietic acid was found to decrease the expression of MMP-2 and u-PA in a concentration-dependent manner . To investigate the possible
mechanisms involved in these events, we performed western blot analysis to find that Abietic acid inhibited phosphorylation of Src. A treatment with Abietic acid to B16F10 cells also inhibited the activation of c-Jun as shown by Western blot and electrophoretic mobility shift assay (EMSA). Finally, Abietic acid were evidenced by its inhibition on the metastasis and tumor volume of B16F10 cells in vivo. Taken together, these findings suggested that Abietic acid could reduce the metastasis of murin melanoma cells, thereby constituting an adjuvant treatment for metastasis control.

並列關鍵字

Abietic acid ； melanoma ； tail vein

參考文獻

Yata N (2008). [Diagnosis and treatment for extra-mammary Paget disease]. Gan To Kagaku Ryoho 35: 596-8.

Tan N，Kaloga M，Radtke O A，et al. Abietane diterpenoids and triterpenic acids from salvia cilicica and their antileishmanial activities [J]. Phytochemistry，2002，61：881–884.

González MA, Pérez-Guaita D, Correa-Royero J, Zapata B, Agudelo L et al (2010). Synthesis and biological evaluation of abietic acid derivatives. Eur J Med Chem. ;45(2):811-6.

Roberto A S，Schweizer A G，Brigitte M Frey，et al. Antiinflammatory activity of abietic acid，a diterpene isolated from Pimenta racemosa var. grissea [J]. Molecular and Cellular Endocrinology，2003，212（1/2）：41–49.

Takahashia N，Kawadaa T，Gotoa T，et al. Abietic acid activates peroxisome proliferator–activated receptor–γ(PPARγ) in RAW264.7 macrophages and 3T3–L1 adipocytes to regulate gene expression involved in inflammation and lipid metabolism [J]. Febs. Letters，2003，550：190–194.