- 學位論文
探討根皮素對於人類子宮頸癌細胞自我更新和血管生成的影響
The influences of phloretin on self-renewal and angiogenesis in human cervical cancer cells
摘要
根皮素是一種主要從蘋果中提取的二氫查爾酮類黃酮 (dihydrogen chalcone flavonoid)，目前臨床上針對其抗氧化、消炎及抗癌活性已有廣泛地研究，然對於子宮頸癌的自我更新和血管生成作用卻不清楚。根皮素可藉由阻斷細胞週期蛋白和細胞週期蛋白依賴性激酶來阻止腫瘤細胞的生長、激活粒線體介導的細胞死亡來誘導細胞凋亡、抑制GLUT2 mRNA和蛋白，進而阻斷糖酵解的路徑，並可對腫瘤細胞的遷移有抑制作用。本篇為了解根皮素在人類子宮頸癌細胞的影響，選取子宮頸癌細胞株(HeLa、CaSki、SiHa)以及人類臍靜脈內皮細胞(HUVECs)來進行試驗。以primary and secondary sphere formation assay判斷單個細胞在合適的條件培養基中自我更新的能力，並鑑定細胞的幹性；以Western blotting進行分析SiHa子宮頸癌起始細胞內CD44和Sox-2的蛋白質表現；以gelatin zymography assay、MMP-3 activity kit和cathepsin S activity assay kit來觀察MMP-2、MMP-3和組織蛋白酶Ｓ的活性；以管形成試驗(tube formation assay)來評估根皮素的血管生成作用；並以DAPI stain 與AVO stain觀察根皮素是否會誘發細胞凋亡及細胞自噬作用。試驗結果發現在以100 μM的根皮素處理後，有顯著抑制SiHa細胞的ALDH1活性、降低形成子宮頸癌起始細胞內CD44和Sox-2的自我更新特性和幹性信號，以及抑制人臍靜脈內皮細胞的侵襲、MMP-2活性和管形成能力，此外，根皮素並不會誘導細胞凋亡及細胞自噬作用。綜合以上試驗的結果可證明，根皮素會抑制子宮頸癌細胞(SiHa)的自我新生、癌細胞幹性和血管生成的能力，或許可以成為癌症治療的抗癌藥物成分。
並列摘要
Phloretin is a dihydrogen chalcone flavonoid which primarily extracted from apple. Many studies have demonstrated the anti-oxidative activity, anti-inflammatory and anti-cancer activities of phloretin. The role of renewal and angiogenesis is unclear. Phloretin can prevent tumor cell growth by blocking cyclin and cyclin-dependent kinases, activate mitochondrial-mediated cell death to induce apoptosis, inhibit GLUT2 mRNA and protein, and block glycolysis. It also can inhibit the migration of tumor cells. To understand the effect of phloretin on human cervical cancer cells, we selected cervical cancer cell lines (HeLa, CaSki, SiHa) and human umbilical vein endothelial cells (HUVECs) for testing. Use primary and secondary sphere formation assay to determine the ability of individual cells to self-renew in the appropriate conditioned medium and identify the stemness of the cells; use Western blotting to analyze the protein expression of CD44 and Sox-2 in the cervical cancer-initiating cells (SiHa); use gelatin zymography assay, MMP-3 activity kit and cathepsin S activity assay kit to observe the activities of MMP-2, MMP-3 and cathepsin S; use tube formation assay to evaluate angiogenesis effect; and use DAPI and AVO stain to observe whether phloretin induces cell apoptosis and autophagy. The test results found that after treatment with 100 μM phloretin, it significantly inhibited the ALDH1 activity of SiHa cells, reduced the self-renewal properties and stemness signals of CD44 and Sox-2 in the cervical cancer-initiating cells (SiHa), and inhibited the invasion of the Human umbilical vein endothelial cell, MMP-2 activity and tube forming ability. Moreover, phloretin did not induce cell apoptosis and autophagy Based on the results of the above experiments, it can be proved that phloretin can inhibit the self-renewal, cancer cell stemness and angiogenesis of the cervical cancer cells (SiHa), and may become an anti-cancer drug component in cancer treatment.
參考文獻
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