Aspergillus terreus 真菌二次代謝物 Terrein 用以抑制 抗藥性乳癌幹細胞生長之研究

Investigation and utilization of the secondary metabolite Terrein from Aspergillus terreus to suppress the growth of drug-resistant breast cancer stem/initiating cells

林黎華(Li-Hua Lin)

指導教授 ： 吳世雄

共同指導教授 ： 沈家寧(Chia-Ning Shen)

國立臺灣大學/生命科學院/生化科學研究所/碩士(2010年)

https://doi.org/10.6342/NTU.2010.10761

摘要

最近的研究顯示，經過癌症傳統治療後仍存在些許抗藥性乳癌幹細胞，而可能會導致癌症復發。因此最新的乳癌治療策略即是發展出專一有效地抑制乳癌幹細胞的藥物，如用以減少乳癌幹細胞自我更新生長、抑制多重抗藥轉運蛋白功能、或可促進細胞凋亡途徑。一般癌幹細胞會表現多重抗藥性轉運蛋白ABC transporter，如MDR1、MRP3和ABCG2，而可將抗癌藥物，如: mitoxanthrone、gemcitabine、doxorubicin或5-fluorouracil排出細胞外。ABCG2是屬於 ATP-binding cassette transporter (ABC) subfamily G，廣泛表現於幹細胞和癌幹細胞中，事實上，ABCG2又名為breast cancer resistance protein (BCRP)，最早於doxorubicin抗藥性之MCF-7乳癌細胞株被分離出來，已知ABCG2可調控在幹細胞中的吡喀紫質恆定性 (protoporphyrin homeostasis) ，其中可將Hoechst 33342染料外排而形成邊緣族群特性 (side-population) 也又廣泛應用在幹細胞與癌幹細胞的鑑定上。因此研究推測ABCG2 不只會外排有害於生物體之異物，還會參與調控細胞代謝和自我更新能力。
Terrein 是一種真菌代謝產物，最先是由Aspergillus terreus 真菌分離出，但有關Terrein其生物活性的研究極少。本研究目的是探討Terrein是否具有潛力可做為抑制抗藥性乳癌幹細胞之新藥物；而紫杉醇是最有效治療乳癌的藥物，所以在研究中，我們以紫杉醇做為對照藥物。在目前的研究中我們利用Annexin V和propidium iodide 染色方法首次發現低濃度Terrein足以造成MCF-7 乳癌細胞的凋亡和使其生長停滯於G2M生長期。更進一步的利用邊緣族群特性分析(side-population analysis) 發現，Terrein明顯減少具邊緣族群特性的細胞。而利用吡喀紫質外排測定法 (protoporphyrin IX efflux assay) 中發現Terrein也具抑制紫質外排的效果，推測其具抑制乳癌幹細胞活性的功能。總結本研究發現Terrein對具抗藥性乳癌細胞生長具毒殺作用，並推測Terrein也能夠抑制多重抗藥性轉運蛋白ABCG2的功能並且能進一步減低乳癌幹細胞活性並達成抑制乳癌生長的效果，具有潛力可做為抑制抗藥性乳癌幹細胞之新藥物。

關鍵字

乳癌 ； 癌幹細胞 ； 邊緣族群特性 ； ABCG2 ； Terrein ； 二次代謝產物 ； 抗藥性

並列摘要

Recent studies have demonstrated there are cancer stem/initiating cells existed in breast cancer which display drug resistance to many conventional therapies and can cause cancer relapse. Therefore, the most promising strategy for breast cancer therapeutics is to develop methods that can either suppress self-renewal expansion, inhibit the function of multidrug transporters, or to induce apoptosis on cancer stem/initiating cells. Cancer stem/initiating cells expressed ABC transporters such as MDR1, MRP3 and ABCG2 that can efflux anticancer drugs such as mitoxanthrone, gemcitabine, doxorubicin or 5-fluorouracil. ABCG2, belongs to the ATP-binding cassette transporter (ABC) subfamily G, expressed in wide variety of stem cells and cancer stem cells. In fact, ABCG2, also named as breast cancer resistance protein (BCRP), was first discovered from a doxorubicin-resistant MCF-7 breast cancer cell line. ABCG2 is a Hoechst 33342 dye efflux pump that mediates side-population phenotype and also regulates porphyrin homeostasis in stem cells. It has been proposed that ABCG2 not only functions as a xenobiotic pump, but also affect cell metabolsm and self-renewal ability of stem cells and cancer stem cells.
Terrein is a fungal metabolite and was first isolated from Aspergillus terreus. However, relatively little is known about Terrein. The aim of the current work is to determine if Terrein can serve as a potential compound that can target drug-resistant breast cancer initiating cells. In the current work, we used Taxol, the most effective drug for breast cancer, as a comparison. We found for the first time that low concentration of Terrein (1 nM) was sufficient to suppress the expansion of MCF-7 cells. The suppressive effect was either mediated by induction of apoptosis and cause of cell arrest at the G2M phase. We also revealed treatment of Terrein inhibited side-population and protoporphyrin IX (PPIX) efflux which may lead to suppression of the activity of breast cancer stem/initiating cells. In conclusion, Terrein displayed cytotoxicity against drug-resistant breast cancer cells. Moreover, the current work also indicates that Terrein is a potent inhibitor for ABCG2 transporter which can possibly lead to reduction in the activity of cancer stem/initiating cells. We therefore assume Terrein can possibly used as a therapeutics for breast cancer stem cells.

並列關鍵字

breast cancer ； cancer stem/initiating cells ； side population ； ABCG2 ； Terrein ； secondary metabolite ； drug-resistance

參考文獻

1. Visvader JE, Lindeman GJ (2008) Cancer stem cells in solid tumours: accumulating evidence and unresolved questions. Nat Rev Cancer 8: 755-768.

3. Creighton CJ, Li X, Landis M, Dixon JM, Neumeister VM, et al. (2009) Residual breast cancers after conventional therapy display mesenchymal as well as tumor-initiating features. Proc Natl Acad Sci U S A 106: 13820-13825.

4. Phillips TM, McBride WH, Pajonk F (2006) The response of CD24(-/low)/CD44+ breast cancer-initiating cells to radiation. J Natl Cancer Inst 98: 1777-1785.

5. Yu F, Yao H, Zhu P, Zhang X, Pan Q, et al. (2007) let-7 regulates self renewal and tumorigenicity of breast cancer cells. Cell 131: 1109-1123.

6. Li X, Lewis MT, Huang J, Gutierrez C, Osborne CK, et al. (2008) Intrinsic resistance of tumorigenic breast cancer cells to chemotherapy. J Natl Cancer Inst 100: 672-679.