- 學位論文
探討SFN透過降低 Cdc2 和Cyclin B1複合物結合率誘發人類三陰性乳癌細胞細胞週期停滯於G2 / M期
G2 / M Delay in Human Triple-Negative Breast Cancer Cells after Treatment with SFN via Cdc2 / Cyclin B1 Dissociation
摘要
衛生福利部國民健康署最新公佈的國人十大死因中，惡性腫瘤仍是國人十大死因之首，目前治療癌症的藥物在人體中會產生嚴重的副作用，故開發副作用低的抗癌藥物或輔助性抗癌藥物逐漸受到醫學相關研究的重視，近年來許多研究發現天然植物中藥材或食療性素材中具有生物活性的成分同時也有較強的化學預防性功效 (chemopreventive) ，故期望能發展出具有輔助化學治療的抗癌藥物。 本碩士論文所使用的抗癌活性成分為蘿蔔硫素 (Sulforaphane ; SFN) ，蘿蔔硫素內含異硫氰酸酯基，屬於有機硫化合物，當咀嚼富含SFN的植物（十字花科蔬菜，如青花菜和大白菜）後，會產生葡萄糖蘿蔔硫苷 (Glucoraphanin)，並與黑芥子酶 (Myrosinase) 結合而轉化成SFN。 本篇研究探討SFN對於人類三陰性乳腺癌細胞株MDA-MB-231的抗腫瘤活性。我們的研究結果表明蘿蔔硫素具有誘導細胞週期的G2/ M期停滯和抑制細胞生長的能力。在G2 / M期轉換中，Cdc2和Cyclin B1的表現量不論是造成細胞週期停滯或造成有絲分裂的過程都佔重要的因素。在西方墨點法 (Western-blot) 實驗中發現沒有顯著抑制 Cdc2 的表現量，然而卻增加其磷酸化 Cdc2Tyr-15 的表現量。實驗結果發現 SFN 經由抑制 Cdc25c 而降低 Cdc2 的去磷酸化作用使細胞無法進入有絲分裂期，由於 Cdc2 和 Cyclin B1 之複合物會形成 MPF，之後利用免疫共沉澱法 (Co-IP) 分析 Cdc2 / Cyclin B1複合物的關係。我們的結果證明 SFN 誘導的細胞週期 G2 / M 期停滯和抑制 Cdc2 / Cyclin B1 複合物的生成有關。在 Human cell cycle PCR array 的分析中顯示，經過 SFN 的處理後 CDK5R1 以及 SERTAD1 的基因表現有顯著性增加。CDK5R1 和神經細胞凋亡有關，而 SERTAD1 為致癌基因可通過抑制細胞凋亡來促進各種癌症細胞的存活。利用西方墨點法 (Western-blot) 分析顯示經過 SFN 處理會增加 CDK5R1 的表現且同時抑制 SERTAD1 的表現，這個結果證實SFN 所誘導的細胞週期 G2 / M 期停滯和增加 CDK5R1 的表現且同時抑制 SERTAD1 的表現量有關。這些結果證明 SFN 能提供人類三陰性乳癌治療及預防上一個新方向。
並列摘要
Abstract： Ministry of Health and Welfare (MOHW) recently released the top ten causes of death, cancer is still one of the top ten in Taiwan. So the cancer prevention and therapy is the most important now. In recent years, Researchers have found that natural plant materials or biologically active ingredients have strong chemo-preventive effects and may play a role of cancer treatment or therapeutic drugs. The Sulforaphane (SFN) is one of the isothiocyanate groupswithorganosulfur compounds which were from cruciferous vegetables such as broccoli or cabbages. It is produced when the myrosinase transforms glucoraphanin, a glucosinolate, to SFN upon damage to the plant, such as from chewing. This study investigated the anti-tumor activity of sulforaphane (SFN) in the human triple-negative breast cancer cell line, MDA-MB-231. Our results demonstrate the ability of SFN to induce cell cycle G2 / M arrest and inhibit cell growth. The activity of Cdc2 and Cyclin B1 expression at the G2 / M transition is an important factor in determining whether cell cycle arrest occurs before or during mitotic progression. The results of western blot analysis did not reveal significant inhibition of Cdc2; however, an increase in phosphorylation Cdc2Tyr15 expression was observed. Results show that SFN blocks cell mitosis via the inhibition of Cdc25c activity resulting in a decrease in Cdc2 dephosphorylation. This makes it possible for Cdc2 and Cyclin B1 complex to form the maturation-promoting factor (MPF). In this study, Co-Immunoprecipitation (Co-IP) was used to detect the Cdc2 / Cyclin B1 complex association. Our results revealed SFN-induced cell cycle G2 / M Phase delay through the inhibition of activity related to the Cdc2 / Cyclin B1 association. The results of Human cell cycle PCR array analysis revealed a significant increase in CDK5R1 and SERTAD1 gene expression following treatment with SFN. CDK5R1 has been linked to neuronal apoptosis, whereas SERTAD1 is an oncogene shown to promote the survival of various types of cancer cells through the inhibition of cell apoptosis.Western blot analysis revealed that treatment with SFN inhibits SERTAD1 expression and increases the expression of CDK5R1. This is a clear indication that the SFN-induced cell cycle G2 / M Phase delay may be associated with an increase in CDK5R1 and a decrease SERTAD1 expression. These results demonstrate the potential of SFN as an alternative route for the treatment and/or prevention of breast cancer.