五羥黃酮抑制人類骨肉瘤細胞轉移和侵襲之機制探討

Study of the inhibitory effects of Tricetin on the migration and invasion of osteosarcoma cells

羅方政(Fang-Cheng Lo)

指導教授 ： 楊順發

中山醫學大學/醫學院/醫學研究所/碩士(2014年)

https://doi.org/10.6834/CSMU.2014.00183

摘要

骨肉瘤是惡性程度最高且最常見的原發性惡性骨腫瘤，好發於發育中的兒童及青少年，當發生遠處轉移時，最常轉移之部位為肺部。過去的研究發現基質金屬蛋白水解酶 (Matrix metalloproteinases; MMPs) 中，MMP-9及MMP-2會促進骨肉瘤的轉移並使病人的預後能力變差。黃酮類化合物過去被發現大量存在植物中，具有廣泛的生物學作用，而其中一類化合物－五羥黃酮 (Tricetin) 在許多文獻中被發現具有抑制癌細胞增生，同時誘導癌細胞走向細胞凋亡的效果，包括乳癌及肝癌，但五羥黃酮與骨肉瘤之間的相關性尚未被釐清。因此本實驗擬探討五羥黃酮是否會對骨肉瘤細胞的生長與轉移造成影響。首先本實驗將五羥黃酮劃分為四組濃度 (0、20、40、80 μM)，將其各別加入骨肉瘤細胞株U2OS及HOS中，並利用MTT assay發現五羥黃酮在此濃度差異下，對U2OS與HOS細胞並無毒殺性。透過Zymography assay發現五羥黃酮會顯著抑制U2OS細胞的MMP-9蛋白活性，亦會抑制HOS細胞的MMP-9及MMP-2蛋白活性。利用Wound healing assay及Boyden chamber assay發現五羥黃酮會顯著抑制U2OS與HOS細胞爬行、轉移及侵襲的能力。上述之結果顯示五羥黃酮能抑制此兩株骨肉瘤細胞的轉移，且對U2OS細胞的MMP-9具較專一的抑制效果，因此本實驗想進一步觀察五羥黃酮對U2OS細胞的作用機制。利用Western blot及RT-PCR、Real time PCR發現五羥黃酮能抑制MMP-9的蛋白表現量及MMP-9 mRNA的表現量。Western blot的結果亦顯示五羥黃酮會抑制磷酸化p38及磷酸化Akt訊息傳遞蛋白之表現，並抑制細胞核內NF-κB的蛋白表現量。最後利用p38及Akt抑制劑確認五羥黃酮能透過訊息傳遞路徑p38及Akt來調控下游蛋白表現。總結以上結果，本實驗發現在骨肉瘤細胞中，五羥黃酮能減少細胞核內NF-κB的蛋白表現，並透過調控MMP-9的轉錄及p38、Akt訊息傳遞路徑來抑制MMP-9的表現使癌症轉移能力降低。

關鍵字

五羥黃酮 ； 骨肉瘤 ； 轉移 ； 基質金屬蛋白水解酶

並列摘要

Osteosarcoma is the most malignant and common primary bone tumor, occurring in developing children and adolescents with distant metastasis. The most common site of metastasis is the lungs. Previous studies have revealed that, in matrix metalloproteinases (MMPs), MMP-9 and MMP-2 promote metastasis of osteosarcoma, leading to poor prognosis. Numerous studies have demonstrated that an abundance of flavonoids, which produce a wide range of biological effects, exist in plants. Researchers have also discovered that tricetin, a class of flavonoids, inhibits cancer-cell proliferation and induces apoptosis in cancer cells, including breast and liver cancer cells. However, the correlation between tricetin and osteosarcoma remains unclear. Therefore, we aimed to determine whether tricetin affects the growth and migration of osteosarcoma cells. First, we used various concentrations of tricetin (0, 20, 40, 80 μM) to treat human osteosarcoma cell lines (U2OS and HOS). The cytotoxic effects of tricetin at various concentrations on U2OS and HOS cells indicated that tricetin did not alter U2OS and HOS cell viability. Zymography analysis indicated that tricetin significantly inhibited the protein levels of MMP-9 in U2OS cells, as well as the protein levels of MMP-9 and MMP-2 in HOS cells in a dose-dependent manner. The results of wound healing analysis and Boyden chamber analysis indicated that tricetin significantly reduced mobility, migration, and invasion of U2OS and HOS cells in a dose-dependent manner. We then further examined the effects of tricetin on U2OS cells by using a western blot analysis, reverse-transcription polymerase chain reaction (PCR), and real-time PCR. The results indicated that tricetin reduced protein levels of MMP-9 and MMP-9 mRNA. The results of the western blot analysis also demonstrated that tricetin can inhibit the signal pathway proteins phosphor-p38 and phospho-Akt in U2OS cells, as well as the levels of NF-κB in the nucleus of U2OS cells. Finally, by using p38 and Akt inhibitors, we confirmed that tricetin can regulate downstream proteins in the p38 and Akt signal pathways. Based on these findings, we concluded that tricetin can inhibit the protein levels of NF-κB in the nucleus of U2OS cells, and that tricetin inhibits the migration and invasion of U2OS cells through transcription of the MMP-9 gene and the activities of the p38 and Akt signal pathways, decreasing the protein levels of MMP-9.

並列關鍵字

Tricetin ； osteosarcoma ； metastasis ； matrix metalloproteinases

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