山奈酚抑制腎癌細胞侵襲及移動能力之機轉探討

Study of the Inhibitory Effect and its Mechanism of Kaempferol on Renal Cancer Invasion and Migration

吳旭鎮(Hsu-Chen Wu)

指導教授 ： 張浤榮

中山醫學大學/醫學院/醫學研究所/碩士(2013年)

https://doi.org/10.6834/CSMU.2013.00078

摘要

研究目的：癌症一直以來都位居國人十大死亡原因之第一位，其主要原因與癌細胞轉移(metastasis)的複雜過程使得治療困難度提昇而導致癌症病人死亡。近年來天然植物性黃酮類化合物逐漸受到醫學界的重視並被廣泛研究，且已被證實在抑制乳癌及前列腺癌細胞的增生及誘發細胞凋亡有相當的效果。因此希望藉由本研究，能對於造成腎癌細胞的轉移及侵襲的機制，有較多的了解並能找到可能的輔助治療方法。
研究方法及資料：山奈酚(kaempferol)是一種常見於植物的類黃酮天然化合物，許多的藥理性作用已逐漸被發現，其中包括:抗發炎、抗氧化以及抗癌的效果。因此，針對腎癌細胞(786-O cells)，我們使用不同濃度(0μM-100μM)的山奈酚，來探討對於抑制腎癌細胞增生及侵襲轉移的效果。
研究結果：結果發現，山奈酚在0μM到100μM 的濃度下，對於癌細胞的存活並無顯著的抑制效果，但對於腎癌細胞的侵襲/移動(invasion/migration)能力，則隨著濃度上升而有顯著的抑制效果(P<0.05)。以 gelatin zymography 及西方墨點法的分析結果，顯示 kaempferol 對於腎癌細胞的基質金屬蛋白酵素 matrix metalloproteinase (MMP)-2之酵素活性有顯著抑制的效果，且其抑制效果與kaempferol的濃度成正相關。進一步探討 kaempferol 抑制 MMP-2之酵素活性的可能分子機轉，發現kaempferol 會造成 Akt 以及focal adhesion kinase(FAK)的磷酸化下降。另外在動物實驗中，我們在免疫力不全的老鼠的尾部注入RCC 786-O 細胞，並且給予老鼠餵食不同濃度的kaempferol，在150天後發現治療組老鼠肺部轉移的腫瘤的重量有明顯下降，其肺臟重量僅約為對照組的1/5到1/2倍。
結論與建議：綜合上述結果發現，kaempferol能透過抑制MMP-2及FAK的活性來達到有效抑制腎癌細胞的侵襲及轉移，在未來或許可作為治療腎癌以及抑制腎癌轉移的輔助療法。

關鍵字

腎細胞癌 ； 類黃酮 ； 山奈酚 ； 基質金屬蛋白酵素

並列摘要

Objective：Kaempferol, isolated from several natural plants, is a polyphenol belonging to the subgroup of flavonoids. It possesses various pharmacological activities which include anti-inflammatory, anti-oxidant, anti-microbial, and anti-cancer activities. From this study, we try to figure out the possible underlying mechanisms of metastasis of renal cell carcinoma and to fine out the possible adjuvant treatment of renal cell carcinoma.
Methods and Materials：RCC 786-O cell, a human embryonic kidney cell line, was cultured in certain medium. To determine the cytotoxicity of kaempferol, a MTT colorimetric assay was performed. We also examine the effects of different concentrations of kaempferol on wound healing and cell invasion and migration of 786-O RCC. Gelatin zymography and Western blot were performed to evaluate the change of several proteins, including MMP-2, Akt, FAK, and so on. Animal study was also performed by SCID mice with injection of RCC 786-O cell via tail vein. The one-way analysis of variance was used to test the statistical significances of diffenrence throughtout this study.
Results：Kaempferol can inhibit the invasion and migration of 786-O renal cell carcinoma (RCC) in a dose-dependent manner without cytotoxicity (0μM to 100μM). We also examine the potential underlying mechanisms for its anti-invasive activities on 786-O RCC cells. Gelatin zymography and Western blot showed that kaempferol attenuates the manifestation of metalloproteinase-2 (MMP-2) protein in a concentration-dependent manner (P<0.05). The inhibitive effect on MMP-2 of kaempferol may result from the down-regulation of the phosphorylation of Akt protein as well as focal adhesion kinase (FAK). From our in vivo study with SCID mice model, we can also figure out that kaempferol could safely inhibit 786-O RCC cells’ metastasis to lung with up to 2~5-fold reduction of lung weight among the groups of mice with pretreatment of 2.0mg/Kg and 10.0mg/Kg kaempferol.
Conclusion and Suggestion： The data from our study suggested that kaempferol could in vitro inhibit invasion and migration of RCC 786-O cell through down-regulating MMP-2 manifestation by inhibiting phosphorylation of FAK and Akt. In vivo, kaempferol could exert an anti-proliferative effect on RCC 786-O cell. Therefore, kaempferol might have a potential role in prevention and treatment of metastatic RCC.

並列關鍵字

renal cell carcinoma (RCC) ； flavonoid ； kaempferol ； metalloproteinase (MMP)

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