百里醌對於腎臟癌細胞的轉移與細胞骨架的影響之探討

Studies of effects of thymoquinone on cancer metastasis and cytoskeleton rearrangements in renal cancer cells

林淑文(Shu-Wen Lin 林淑文)

指導教授 ： 謝易修

中山醫學大學/醫學院/生化暨生物科技研究所/碩士(2016年)

摘要

百里醌(TQ)是黑種草油(Nigella sativa)的主要成分。過去研究發現，百里醌在不同的癌細胞株有抑制生長之作用，但其是否會影響腎臟癌細胞的轉移目前還不清楚。而在本實驗中，我們要利用百里醌探討其對於786-O-SI3此腎細胞癌細胞株的轉移(migration)和侵襲(invision)能力之影響、以及其作用轉制。腎臟癌在早期可以毫無症狀，一直到腫瘤太大而壓迫到其它器官或是已轉移才出現症狀，而癌症也隨著其轉移能力越強死亡的機率也就越高。本實驗會先以 MTT assay 檢測百里醌濃度0 μM、5 μM、10 μM、20 μM 對其腎臟癌細胞有無毒殺作用。接著再以細胞侵襲實驗(Boyden chamber invasion assay)、細胞移動實驗(Boyden chamber Migration assay)以及傷口癒合實驗(wound healing assay)去觀察 786-O-SI3 的細胞侵襲以及細胞移動的能力。在實驗的結果下證實0 μM - 20 μM 的百里醌對 786-O-SI3並無毒殺作用，且低濃度 10 μM 及20 μM的百里醌，在 24 小時處理之下，786-O-SI3 細胞侵襲(invasion)以及轉移(migration)能力有明顯被抑制的現象。再以 gelatin zymography assay 去觀察投予百里醌濃度 0 - 20 μM 的 786-O-SI3 其 MMP-2 以及 u-PA 的活性有顯著被抑制的現象。以 cell-matrix 貼附能力實驗方法，證實隨著百里醌濃度的提升 786-O-SI3 貼附於 collagen (type I and IV)能力有下降的趨勢。這些結果都說明了百里醌會去抑制 786-O-SI3 爬行轉移的能力。所以我們進而使用西方墨點法(Western blot)去觀察 786-O-SI3 在加藥處理後其蛋白質的變化，實驗結果隨百里醌濃度的上升其p-ERK、p-Src、p-paxilin 178、PI3K有顯著被抑制的現象。最後，我們使用10 ng/mL 的 TGF-β 去誘導 786-O-SI3 使其細胞間質化後，利用細胞移動實驗(Boyden chamber Migration assay)、免疫螢光染色以及gelatin zymography assay 等分析方法，去觀察百里醌對786-O-SI3的影響，結果也顯示由 TGF-β 所誘導 786-O-SI3 細胞轉移(migration)、侵襲(invasion)能力以及細胞間質化的作用有明顯被抑制的現象，且 MMP-2 以及 u-PA 活性有顯著被抑制的現象。因此，在抑制腎臟癌細胞轉移以及侵襲，百里醌具有潛在的治療效果。

關鍵字

百里醌腎細胞癌 ； 侵襲 ； 轉移 ； MMP-2 ； u-PA ； 細胞間質化

並列摘要

Thymoquinone (TQ) is the major active component of the volatile oil of Nigella stativa seeds. Previous studies showed that TQ can inhibit various of cancer cell growth, but its mechanism is not yet clear. The aim of this study was to investigate the possible effects of TQ in inhibit on the invasion and migration of 786-0-SI3 renal cell carcinoma (RCC) cells. Kidney cancer (renal cell cancer) signs and symptoms, including include blood in the urine, pain, fever, weight loss, etc. It is noteworthy that kidney cancer may have no symptoms at an early stage, until the tumor is too large and oppression have been transferred to other organs or cancer cell metastasis to other organ, the stronger the higher the probability of death.In this study, we use MTT assay to detect that dose-dependent (0 μM, 5 μM, 10 μM and 20 μM) cytotoxicity effects of TQ on renal cell carcinoma cell lines 786-0-SI3.Thne 786-0-SI3 cell lines was treated with TQ up to 0 μM, 5 μM, 10 μM and 20 μM for a defined period and then subjected to Boyden chamber invasion assay, Boyden chamber motility assay, wound healing assay, gelatin zymography assay, and cell adhered to collagen (type I and IV) assay examine the impacts of TQ. The result was showed significant in inhibition of cell invasion and migration, and TQ decreased matrix metalloproteinases-2 (MMP-2) and u-PA. In Western blot, we found that TQ decreased p-ERK, p-Src, p-paxillin 178 and PI3K. Final, we treated with TGF-β (10 ng/mL) to induced 786-0-SI3 cell line epithelial-mesenchymal transition (EMT), then used Boyden chamber migration assay, Immunofluorescence staining and gelatin zymography assay to the impacts of TQ in 786-O-SI3 cell lines. The result was showed significant in inhibition of cell invasion and migration, and TQ decreased MMP-2 and u-PA. In conclusion, TQ inhibited the invasion of renal cancer cells and may have potential use as a chemopreventive agent against RCC metastasis.

並列關鍵字

Thymoquinone ； renal cell carcinoma ； MMP-2 ； u-PA ； EMT ； invasion ； migration

參考文獻

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