Deoxyelephantopin衍生物抑制人類三陰性乳腺癌之研究

Research on Inhibitory Effect of Deoxyelephantopin Derivatives on Human MDA-MB-231 Triple-Negative Breast Cancer Cells

陳若羽(Jo-Yu Chen)

指導教授 ： 侯文琪

共同指導教授 ： 徐麗芬

臺北醫學大學/藥學院/生藥學研究所/碩士(2014年)

摘要

三陰性乳癌(TNBC)為一種高度轉移性乳腺癌，目前化學療法是唯一適用於全身性的治療方式。然而，持續使用化療藥物會產生抗藥性導致治療上的困難。因此，具有生物活性的草藥或其衍生物單獨或與抗癌藥物合併使用也許能提供乳癌患者另一種有效的治療方式。在本研究中，我們從菊科藥用植物地膽草中分離主要活性之化合物deoxyelephantopin (DET)，並半有機合成新的衍生物D35，來研究DET與D35對抑制人類三陰性乳癌細胞的效果。藉由體外細胞增生活性檢測、即時攝影、Boyden chamber及免疫組織化學分析，我們發現在MDA-MB-231細胞的遷移、入侵等活動力上，D35比DET有更好的抑制效果(有效劑量降低2~3.6倍)。在體內小鼠肺轉移模型研究中顯示，以D35預處理組的小鼠(preD35-10, 10 mg/kg BW)顯著降低MDA-MB-231細胞轉移到肺的數目達到83% 抑制效果，而DET-10 (10 mg/kg BW)、D35-10 (10 mg/kg BW)和PTX-5 (paclitaxel, 5 mg/kg BW)的抑制效果分別為38%、50%、62%。此外，D35與化療藥PTX交替給藥處理(PTX 5 mg/kg + D35 2 mg/kg BW)並可顯著抑制71% 癌細胞轉移到肺臟組織。流式細胞儀試驗結果顯示，經48小時處理之後，D35、DET可以分別造成21.4%、19.3% 的癌細胞凋亡，而PTX主要造成癌細胞增生停滯於G2/M。而西方墨點與免疫組織化學染色分析結果顯示，證明D35的處理能顯著抑制MDA-MB-231細胞中生物指標蛋白質如VEGF、Ki67、COX-2、E-cadherin、N-cadherin和snail之表現量。綜合目前研究成果顯示，新穎的倍半萜衍生物D35具有進一步發展成為化療藥物之輔助劑或增敏劑的潛力。

關鍵字

三陰性乳腺癌 ； deoxyelephantopin (DET) ； 衍生物 ； 癌細胞轉移

並列摘要

Triple-negative breast cancer (TNBC) is a highly metastatic breast cancer type, and chemotherapy is the current consideration for systemic therapy for the disease. However, drug resistance and adverse side-effects to chemotherapeutics in breast cancer patients are commonly observed. The use of bioactive phytoagent or its derivative, alone or in combination with anti-cancer drug, may provide alternative and promising treatment options. In this study, we isolated a bioactive sesquiterpene lactone (SL) deoxyelephantopin (DET) from medicinal plant Elephantopus scaber (Asteraceae) and semi-organically synthesized its derivative (designated D35) through collaboration, and investigated DET and D35 effects on inhibiting human TNBC cell MDA-MB-231 in vitro and in xenograft lung metastasis mouse model. By employing a number of bioassays, including cell viability assay, Boyden chamber assay, time-lapse microscopy, and immunohistochemistry assay, we observed that D35 exhibited a better effect (2~3.6-fold decreased in effective concentrations) than DET on inhibiting migration, invasion, and motility of MDA-MB-231 cells in a concentration-dependent manner. The in vivo data showed that pretreatment with D35 (preD35-10, 10 mg/kg BW) intraperitoneally (i.p.) before tail-vein inoculation of MDA-MB-231 cancer cells significantly reduced the number of metastatic pulmonary tumor foci (83%) in mice, and the suppressed effects of post-treatment with DET-10 (DET, 10 mg/kg BW), D35-10 (D35, 10 mg/kg BW), and PTX-5 (paclitaxel, 5 mg/kg BW) were shown as 38%, 50%, and 62%, respectively. Notably, PTX-5+D35-2 combinational group by alternate administration of PTX-5 and D35-2 (D35, 2 mg/kg BW) greatly reduced lung metastasis
of MDA-MB-231 to 71%. Flow cytometry results showed that, after a 48-h treatment, D35 and DET resulted in 21.4% and 19.3% cancer cell apoptosis, and PTX mainly caused cell-cycle arrest at G2/M phase. According to the results from western blot analysis and immunohistochemistry, D35 significantly inhibited several key biomarkers, such as VEGF, Ki67, COX-2, N-cadherin and snail in MDA-MB-231 cells or tumors. Overall, our results suggest that the novel plant SL derivative, D35, may have a potential to further development into a complementary or sensitizing agent to chemotherapeutic drug PTX.

並列關鍵字

Triple-negative breast cancer (TNBC) ； deoxyelephantopin (DET) ； derivative ； cell metastasis

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