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| 題名: | 由土曲黴、錫蘭七指蕨與番荔枝乙醯合成物篩選乳癌起始細胞標靶活性化合物
Active components of A. terrus, H. zeylanica, and Annonaceous Acetogenins against ABCG2-expressing breast cancer initiating cells |
| 作者: | 廖紋瑩;Liao, Wen-Ying |
| 貢獻者: | 藥物化學研究所博士班 |
| 關鍵詞: | 乳癌起始細胞;轉運蛋白ABCG2;轉錄因子Nanog;土曲黴;錫蘭七指蕨;番荔枝乙醯合成物;Breast tumor initiating cells;ABCG2 transporter;Nanog;Terrein;uognins;Annoaceous Acetogenins |
| 日期: | 2012-06-25 |
| 上傳時間: | 2012-08-31 16:39:31 (UTC+8) |
| 出版者: | 中國醫藥大學 |
| 摘要: | 近年來研究指出，乳癌起始細胞(Breast tumor initiating cells, 又名乳癌幹細胞 (breast cancer stem cells)) 具有高度的致腫瘤生成與自我更新的能力，是導致治療失敗或是癌症復發的主因。本研究欲尋找能專一有效地抑制乳癌起始細胞的天然活性化合物，來建構新的乳癌治療策略。已知具抗藥性乳癌細胞或是乳癌起始細胞表現ABCG2，轉運蛋白，而最近研究指出ABCG2在胚幹細胞(embryonic stem cells)中的重要生理功能之一是調控細胞內紫質的恆定性 (porphyrin homeostasis)，而當ABCG2的功能被抑制而紫質恆定被破壞時，胚胎幹細胞自我更新能力也會受影響。在本論文也發現ABCG2參與調控乳癌起始細胞內紫質的恆定性，因此我利用ABCG2會專一排出紫質(porphyrin efflux)特性來發展藥物篩選方法，分別從真菌 (Aspergillus terreus及Malbranchea sulfurea)、錫蘭七指蕨 (Helminhtostachys zeylanica)中、倒手香 (Plectranthus amboinicu) 篩選、番荔枝科 (Annonaceae) 植物之乙醯生合物 (Annoaceous acetogenins)中，尋找可抑制ABCG2功能的天然活性化合物，再進一步探討這些化合物能否專一有效地抑制乳癌起始細胞致腫瘤生成與自我更新的能力。
由土曲黴 (Aspergillus terreus)的二次代謝物中，發現Terrein可抑制ABCG2外排紫質的功能，針對高度表現ABCG2的乳癌細胞株具有毒殺性，其機制是經由活化caspase-7導致乳癌細胞的凋亡，以及透過抑制PI3K/Akt訊息傳遞途徑減少細胞增生，並降低ABCG2轉移到細胞膜上。 從錫蘭七指蕨裡，發現有十五種黃酮類化合物(ugonins)具有不同程度抑制ABCG2外排紫質的功能，其中ugonin J不僅在動物體內有效的降低腫瘤生成，藉由抑制ABCG2的功能，破壞紫質的恆定，導致p53的活化進而減少Nanog的表現，抑制乳癌起始細胞的自我更新生長及形成乳腺球結構 (mammosphere)的能力。另外，從番荔枝科植物的乙醯生合物分析發現，雖然乙醯生合物只有些微抑制ABCG2外排紫質的功能，卻會透過破壞細胞體內鈣離子的恆定，由核磁共振儀 (NMR) 與等溫滴定量熱法 (Isothermal titration calorimetry) 的試驗，進一步闡明乙醯生合物可會與鈣離子形成疏水性複合物而具有穿過細胞膜，透過胞內鈣離子追蹤實驗，發現添加乙醯生合物可增加細胞質及粒線體內鈣離子，導致粒線體膜電位去極化，造成細胞凋亡。
總結本研究的結果，我們發現ABCG2參與調控乳癌起始細胞內紫質的恆定性及自我更新生長扮演重要角色，在尋找能專一有效地抑制乳癌起始細胞的天然活性化合物的過程中，發現Terrein透過抑制PI3K/Akt訊息傳遞途徑降低ABCG2轉移到細胞膜上及導致乳癌細胞的凋亡；ugonin J可透過抑制ABCG2並降低Nanog表現，進而減少乳癌起始細胞的自我更新及生長。在化合物篩選中，我們也發現乙醯生合物的生物活性機制是透過破壞細胞體內鈣離子的恆定，未來預期可做為癌細胞的選擇性毒殺劑。
Recently studies demonstrated that breast tumor-initiating cells (BTICs), also named breast cancer stem cells (CSCs), can cause cancer relapse due to highly tumorigenic and possess the capacity to self-renewal, and display multidrug resistance to many current therapies. Therefore, a suitable therapeutic strategy for breast cancer is to develop new method for searching natural bioactive compounds that can target cell apoptosis and survival pathway of BTICs. Breast drug-resistant or tumor-initating cells expressed ABCG2, ATP-binding cassette (ABC) transporter. Previous study displayed the inhibition of ABCG2 led to disruption of porphyrin homeostasis and affected the self-renewal ability in embryonic stem cells. We initially found that the ABCG2-expressing subpopulation of breast cells is able to efflux protoporphyrin IX (PPIX). Establishing and utilizing a porphyrin-efflux assay, we screened for 13 compounds from Aspergillus terreus, 4 compounds from Malbranchea sulfurea, 15 natural products from the rhizomes of Helminthostachys zeylanica, 2 compounds from Plectranthus amboinicu, and 8 compounds were isolated from the Formosan Annonaceous plants included Annona muricata, A. montana, A. squamosa, A. reticulata, and Rollinia mucosa. Furthermore, to investigate if those bioactive compounds could effective inhibit the tumorigencity and self-renewal ability of BTICs.
At first, we revealed Terrein isolated from A. Terreus had strong cytotoxic potency against ABCG2-expressing breast cancer cells. And we further found that the ability of Terrein to reduce ABCG2-expressing breast cancer cells was possibly mediated by activation of caspase-7 pathway and inhibition of Akt signaling.
Among these flavonoids, ugonins J and K, which has a free-rotational functional group, were found to be able to induce apoptosis in both ABCG2+ and ABCG2- cell populations. We further revealed that treatment with ugonin J significantly reduced the tumorigenic potential of breast cancer MCF-7 cells in vivo and efficiently suppress propagation of the ABCG2-expressing CD24-/lowCD44+ cell subpopulation in mammospheres. This suppression was possibly due to disruption of porphyrin homeostasis which led to p53 activation and Nanog reduction as overexpression of Nanog can counteract the suppressive effect of ugonin J.
In Formosan Annonaceous plants, we discovered a series of acetogenins can slight inhibit the function of ABCG2 transporters. We demonstrate disruption of equilibrium of intracellular calcium levels mediates the cell cytotoxicity and inhibit ABCG2 function. Utilizing NMR and isothermal titration calorimetry, we identified calcium ions were chelated with the hydroxylated tetrahydrofuran rings of acetogenins which resulted in formation of hydrophobic [acetogenins-Ca2+] complexes, which provide a clue to explain why acetogenins can penetrate through cell membrane. Evidences observed by conforcal microscopy further revealed acetogenins treatment disrupted calcium homeostasis and caused mitochondrial depolarization that then mediates cytotoxicity against various cancer cells.
In conclusion, the current work determined the role of ABCG2 in modulation of porphyrin homeostasis in breast cancer cells. Based on functional screening for natural bioactive compounds, we found that Terrein can suppress Akt activity and translocation of ABCG2 which raised the potential of using Terrein to overcome Akt-mediated drug-resistance in cancer cells. The current work also revealed flavonoids with a free-rotational functional group can possibly be utilized to suppress the propagation of breast initiating/stem cancer cells. The current work also identified Annonaceous acetogenins could be used as selective cytotoxic agent to cancer cells disrupted calcium homeostasis. |
| 顯示於類別: | [藥物化學研究所] 博碩士論文
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