- 學位論文
探討鼠尾草酸對黑色素瘤與乳癌之抑制細胞生長作用機轉與增進抗癌藥物之治療效果
Carnosic Acid Impedes Cell Growth and Enhances Anti- Cancer Effects of Anti-cancer Drugs in Melanoma and Breast Cancer Cells
摘要
前言：鼠尾草酸(Carnosic acid, CA)是迷迭香中一種主要的多酚類雙萜，已經被發現有許多功能，包括抗癌活性。 方法：本研究中使用人類乳癌細胞株(MCF-7, ZR-75-1, MDA-MB-231, MDA-MB-453, and BT-474)、纖維母細胞株(NIH-3T3)與黑色素瘤細胞株(B16F10)來測試鼠尾草酸的抗癌作用。在乳癌的部分，利用MTT法、傷痕癒合測試與聚落形成實驗來檢測細胞的生長與增殖，以流式細胞儀測定細胞週期，COX4、GAPDH、BAX、Bcl-2、Bcl-xL、β-actin、cytochrome C (Cyt.C)、endonuclease G (EndoG)與apoptosis inducing factor (AIF)的表現量則以西方墨點法與定量即時聚合酶鏈鎖反應進行測定，ATP生產量的測定使用Mitochondrial ToxGlo assay完成。而在黑色素瘤的部分，除了MTT法、傷痕癒合測試與聚落形成實驗，BrdU法亦被使用來測試黑色素瘤細胞B16F10的細胞生長與增殖。細胞週期仍以流式細胞儀測定，而p21與p27的表現量則以西方墨點法檢測。研究中亦建立黑色素瘤細胞移植動物模型，並以鼠尾草酸、卡莫司汀(carmustine，BCNU)或洛莫司汀(lomustine，CCNU)進行治療與評估。 結果：在乳癌細胞的測試結果中，鼠尾草酸的作用並不會受到動情激素受體陽性或陰性的影響，鼠尾草酸可以調節乳癌細胞的BAX、Bcl-2與Bcl-xL的表現量，啟動BAX的訊息傳導並導致凋亡因子釋放。此外，鼠尾草酸與順鉑(cisplatin)的合併療法對MCF-7 與MDA-MB-231細胞具有提升人類乳癌細胞對順鉑的敏感度的傾向。另一方面，本篇研究亦探討鼠尾草酸對黑色素瘤的作用，發現鼠尾草酸顯著抑制黑色素瘤細胞B16F10的生長，且使其細胞週期停滯，鼠尾草酸可使細胞週期滯留在G0/G1 期並增加p21的表現。再者，鼠尾草酸可增強卡莫司汀與洛莫司汀在B16F10細胞中引發的細胞毒性與細胞週期停滯。在體內測試中，鼠尾草酸抑制腫瘤生長同時也降低麩胺酸苯醋酸轉氨基酵素(aspartate aminotransferase，AST)與血清麩胺酸丙酮酸轉氨基酵素(alanine aminotransferase，ALT)的數值。 結論：鼠尾草酸或許可以作為一種安全且有效的新化療藥物。
並列摘要
Background: Carnosic acid (CA), a major polyphenolic diterpene in Rosmarinus officinalis, has been reported to have multiple functions, including anti-tumor activity. Methods: To evaluate the anti-cancer effects of CA, the human breast cancer cell lines (MCF-7, ZR-75-1, MDA-MB-231, MDA-MB-453, and BT-474), fibroblast cell line (NIH-3T3) and melanoma cell line (B16F10) were used and tested. For the breast cancer cells, MTT assay, wound healing and colony formation were utilized for the detection of cell growth and proliferation. The cell cycle was determined by flow cytometry. COX4, GAPDH, BAX, Bcl-2, Bcl-xL, β-actin, cytochrome c (Cyt.C), endonuclease G (EndoG) and apoptosis inducing factor (AIF) expression were detected by western blotting and quantitative RT-PCR. The ATP production detection was done by Mitochondrial ToxGlo assay. For the melanoma cells, BrdU incorporation was used to detect melanoma cell growth and proliferation in addition to MTT assay, wound healing and colony formation. Flow cytometry was also conducted to detect cell cycle, and western blotting was conducted to detect p21 and p27 expressions. Melanoma cell xenograft model was also established, and the treatment potential of CA, carmustine (BCNU), or lomustine (CCNU) was evaluated in this present study. Results: According to the results of the breast cancer cells experiment, the effect of CA was not affected by estrogen receptor (ER) either positively or negatively. CA was able to regulate the expression of BAX, Bcl-2 and Bcl-xL, trigger BAX translocation which lead to release of apoptotic factors in breast cancer cells. Moreover, CA was found to have a tendency to sensitize ER-positive and -negative human breast cancer cells to cisplatin by combined treatment of CA and cisplatin on MCF-7 and MDA-MB-231 cells. On the other hand, the investigation of the effect of CA on melanoma found that CA exhibited significant growth inhibition and cell cycle arrest in melanoma B16F10 cells. We also found that CA triggered cell cycle arrest at G0/G1 phase and enhanced p21 expression. Moreover, CA could enhance BCNU- and CCNU-mediated cytotoxicity and cell cycle arrest in B16F10 cells. Finally, we found that CA inhibited tumor growth, and reduced the values of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in vivo. Conclusion: CA may be safe and useful as a novel chemotherapeutic agent.