臺灣博碩士論文加值系統

癌症治療藥物的副作用及癌症細胞的抗藥性是造成現今仍無法有效治療癌症的重要因素，而找出使癌細胞提高對於治療藥物敏感性的方法及其機制是當今一項熱門的研究議題。在Her2陽性的乳癌患者中，對癌賀平(為Her2的單株抗體，透過直接抑制Her2所介導的訊息傳遞而使細胞凋亡)產生抗藥性而使癌症復發並產生更強烈的抗藥性是其主要的治療障礙。在本篇研究中，我們將探討合併使用靈芝多醣體和臨床乳癌標靶藥物賀癌平在賀癌平敏感型的BT474及具賀癌平抗性的SKBR3人類乳癌細胞中的治療效果。我們的研究顯示，相較於單獨給予一種藥物，同時投與靈芝多醣體和標靶藥物賀癌平，不管在賀癌平敏感型(BT474)或是具有抗性(SKBR3)的人類乳癌細胞株中，均可以更有效的降低癌細胞的存活率。在合併使用靈芝多醣體和賀癌平的情形下，其細胞凋亡的情形會更加明顯。接下來，為了確認靈芝多醣體是透過什麼機制來促進乳癌細胞對於賀癌平的敏感性，因此我們首先由Her2總表現量來探討，而結果顯示在BT474及SKBR3細胞中靈芝多醣體會透過減少AKT的磷酸化，進一步透過TWIST及YB-1去調控Her2的表現量。此外，IGF1R為目前已證實和賀癌平抗性相關的一主要調控因子，為了探討靈芝多醣體是如何降低SKBR3對賀癌平的抗性，我們由IGF1R的表現量來解釋。總的來說，我們認為靈芝多醣體與臨床乳癌標靶藥物賀癌平合併用藥的治療方式或許可以在改善具賀癌平抗藥性的乳癌治療上成為一個有效的策略。

Resistance and side-effect of clinical drugs are crucial factors that limited its usage. That why sensitization of cancer cell to drug is a hot issue now. Trastuzumab resistance cause relapse of the tumor or dosing mass anti-cancer drug is a major obstacle in the treatment of patients with HER2-positive breast cancer patients. In this study, we explored the combined effects of combining traditional Chinese medicine, fucose-containing fraction of Reishi (FFOR) and breast cancer target drug trastuzumab, on BT474 trastuzumab sensitive breast cancer cell line and SKBR3 trastuzumab resistance breast cancer cell line. Our results showed that combining FFOR and trastuzumab, which binds to HER2 with high affinity, inducing a cytostatic effect associated with G1 arrest, reduction in cell proliferation, and apoptosis via up-regulation of the cyclin-dependent kinase inhibitor p27kip1, was more effective in killing BT474 and SKBR3 human breast cancer cells than either FFOR or trastuzumab single. The combination of FFOR and trastuzumab was synergistic induced apoptosis, causing more cell death than any single treatment in both BT474 and SKBR3. Next, to clarify the mechanism why the sensitization to trastuzumab was enhance in breast cancer cells, we investigated the expression of Her2. We show that FFOR reduced phosphorylation of AKT then can regulate the expression of Her2/neu through a pathway that involves TWIST and YB-1 in BT474 and SKBR3 cells. Furthermore, IGF1R signaling implicated to trastuzumab resistance has displayed. We also observed that the expression of IGF1R was reduced after FFOR treatment in SKBR3. In summary, the present study reveals that a combination of FFOR and trastuzumab may be a effectively strategy for improving the resistance of trastuzumab.

Contents----------------------------------------1
中文摘要-----------------------------------------3
Abstract----------------------------------------4
Introduction------------------------------------5
Materials and Methods---------------------------8
Cell culture------------------------------------8
Preparation of FFOR-----------------------------8
MTT Assay---------------------------------------8
Cell counting-----------------------------------8
Medium-Effect Analysis of Combine Effect--------9
Cell extracts-----------------------------------9
Antibody----------------------------------------9
Western Blotting Analysis-----------------------10
Apoptosis assay---------------------------------10
Cell cycle assay--------------------------------10
Result ----------------------------------------11
FFOR inhibits the proliferation of breast cancer cells, BT474 and SKBR3 --------------------------------11
BT474 human breast cancer cell was sensitized to anti-cancer drug trastuzumab by FFOR ----------------11
SKBR3 human breast cancer cell was also sensitized to anti-cancer drug trastuzumab by FFOR ----------------12
Trastuzumab cooperates with FFOR increased cell death phenomenon caused by trastuzumab in BT474 human breast cancer cell line--------------------------------13
Trastuzumab combined treatment with FFOR increased cell death phenomenon caused by FFOR in SKBR3 human breast cancer cell line--------------------------------13
FFOR induces cell cycle arrest at G0-G1 phase in BT474 and SKBR3 breast cancer cells-----------------------14
FFOR reduced expression of ILK, Twist, YB-1 and Her2 in BT474 and SKBR3---------------------------------14
FFOR regulates the IGF1R pathway inducing SKBR3 sensitivity to trastuzumab----------------------------------15
Discussion--------------------------------------16
Reference---------------------------------------20
Figure legend-----------------------------------24
Figure1. FFOR inhibits the proliferation of breast cancer cells, BT474 and SKBR3--------------------------25
Figure2. BT474 human breast cancer cell weas sensitized to anti-cancer drug trastuzumab by FFOR------------28
Figure3. SKBR3 human breast cancer cell was also sensitized to anti-cancer drug trastuzumab by FFOR---------32
Figure4. Trastuzumab cooperates with FFOR increased cell death phenomenon caused by trastuzunab in BT474 human breast cancer cell line-------------------------35
Figure5. Trastuzumab combined treatment with FFOR increased cell death phenomenon caused by FFOR in SKBR3 human breast cancer cell line--------------------------------37
Figure6. FFOR induces cell cycle arrest at G0-G1 phase in SKBR3 and BT474 breast cancer cells.------------40
Figure7. FFOR reduced expression of ILK, Twist and Her2 in BT474 and SKBR3---------------------------------43
Figure8. FFOR regulates the IGF1R pathway inducing SKBR3 sensitivity to trastuzumab----------------------44

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