台灣檜木醇抑制乳腺癌幹細胞自我更新及其機轉之研究

中山醫學大學機構典藏 CSMUIR > 醫學院 > 生化微生物免疫研究所 > 博碩士論文 > Item 310902500/20877

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Title:	台灣檜木醇抑制乳腺癌幹細胞自我更新及其機轉之研究 Hinokitiol suppresses the self renewal of breast cancer stem/progenitor cells and the mechanism
Authors:	陳仕明 Chen, Shih-Ming
Contributors:	中山醫學大學:生化微生物免疫研究所;許立松;張文瑋
Keywords:	台灣檜木醇;乳癌幹細胞;miR-494-3p;梳複合蛋白BMI;miR-145-5p;細胞自噬 Hinokitiol;breast cancer stem cell;miR-494-3p;Polycomb complex protein BMI1;miR-145-5p;Autophagy
Date:	2019
Issue Date:	2020-07-03T09:06:57Z (UTC)
Abstract:	乳腺癌不僅是世界上女性惡性腫瘤發生率排名第一，也是台灣女性最常見的癌症。癌症幹細胞屬於癌症細胞中的亞群，其對於癌症的開始、治療的抗藥性、和腫瘤轉移都有相當重要的關聯。而癌症幹細胞在癌症治療上是很重要的靶點。乳癌幹細胞和其他癌症幹細胞一樣有特殊的細胞表面標記CD24-CD44+及高乙醛脫氫酶活性，且具有形成腫瘤球體的能力，這些都可以幫助監測癌症幹細胞的活性。台灣檜木醇是從台灣檜木提煉出來的單萜類，除了有抗發炎或是抗微生物感染的效用外，更被發現有抑制癌症細胞的生長。檜木醇可以經由caspase 3 路徑或是造成細胞週期停滯來抑制癌症細胞的凋亡，也可以經由自噬作用導致老鼠乳癌細胞和大腸直腸癌細胞的死亡。由這些資料推論檜木醇可能可以做為嶄新的抗腫瘤化合物，因此我們將檜木醇加入乳癌幹細胞共同培養觀察到可以抑制其自我更新的能力。BMI1屬於polycomb complex蛋白質會控制正常和惡性乳腺細胞的自我更新能力，且在正常乳腺上皮細胞過度表現BMI1會增加其腫瘤球形成能力。我們在加入檜木醇的乳癌幹細胞發現其BMI1蛋白質表現會被下調但是mRNA的表現卻沒有變化。MiRNAs屬於約19-24個核醣核酸大小的非編碼核糖核酸。雖然miR-494-3p在癌症的角色仍屬於有爭論的，但是最近已被證實可以做用在乳癌細胞的PAK1上來抑制細胞的侵襲性。我們的研究發現檜木醇可以在乳癌幹細胞腫瘤球體誘發miR-494-3p的表現，而抑制miR-494-3p可以經由作用在BMI1消除檜木醇取消其抑制作用的效果。在人類乳癌細胞上過度表現miR-494-3p會抑制乳癌腫瘤幹細胞的生長，同時在兩個獨立公開的資料庫可以發現miR-494-3p的過度表現強烈的和病人生存呈負相關。在異種移植乳癌細胞的動物上可以發現檜木醇可以經由上調miR-494-3p和下調BMI1的表現來抑制腫瘤的生長。而台灣檜木醇可抑制三陰性乳癌細胞形成癌症球體的能力，自噬作用抑制劑chloroquine的處理則可減低檜木醇的抑制效果，而檜木醇可誘導三陰性乳癌細胞內LC3-II含量增高，以及抑制細胞內Akt3表現，這些結果顯示檜木醇可誘導三陰性乳癌細胞產生自噬作用。但我們卻發現，同時抑制miR-494-3p無法減弱檜木醇抑制功效，而檜木醇處理的三陰性乳癌細胞中則觀察到miR-145-5p的增加，抑制miR-145-5p則能削減檜木醇對三陰性乳癌的抑制作用，而過度表現miR-145-5p亦可直接抑制三陰性乳癌細胞的自我更新，並過度表現miR-145-5p可抑制三陰性乳癌細胞內Akt3及增加LC3-II表現。總結這些研究結果我們發現檜木醇可以同是在體外和體內經由上調miR-494-3p來抑制BMI1的表現來達到抑制乳癌幹細胞的活性。 Breast cancer has not only been the most prevalent cancer in female around the world but also in Taiwan. Although the progress in local treatment (surgery, radiotherapy) and adjuvant therapy have increased survival in early breast cancer, yet almost a fifth of patients will develop local or distant recurrence within 5 years of diagnosis. Hinokitiol (β-thujaplicin) is a tropolone-related compound that has anti-microbe, anti-inflammation, and anti-tumor effects. Cancer stem/progenitor cells (CSCs) are a subpopulation of cancer cells with tumor initiation, chemoresistant, and metastatic properties which play a very important role in the treatment failure of cancer. They also have been considered the important therapeutic target in future cancer therapy. Previous studies reported that hinokitiol exhibits an anti-cancer activity against murine tumor cells through the induction of autophagy. The current research revealed that hinokitiol suppressed the self-renewal capabilities of human breast CSCs (BCSCs) and inhibited the expression of BMI1 at protein level without suppressing its mRNA. Treatment of hinokitiol in mammospheres induced the expression of miR-494-3p and inhibition of miR-494-3p expression in BCSCs. This treatment abolished the suppressive effects of hinokitiol in mammosphere formation and BMI1 expression. BMI1 is a target of miR-494-3p by luciferase-based 3′UTR reporter assay. Overexpression of miR-494-3p in BCSCs caused the down-regulation of BMI1 protein, inhibition of mammosphere forming capability, and suppression of their tumorigenicity. Moreover, miR-494-3p expression was significantly and inversely correlated with patient survival in two independent public database sets. Furthermore, treatment of hinokitiol in vivo suppressed the growth of xenograft human breast tumors as well as the expression of BMI1 and ALDH1A1 in xenograft tumors. We also found that hinokitiol inhibited mammosphere formation capability in triple negative breast cancer (TNBC) cells and the treatment of chloroquine diminished the inhibitory effect of hinokitiol to self-renweal of TNBC cells. The expression of LC3-II in TNBC cells was increased with simultaneously downregulating Akt3 expression. These data suggest that hinokitiol could induce autophagy in TNBC cells and leads to the suppression of self-renewal capability. However, the inhibition of miR-494-3p did not diminished the inhibitory effect of hinokitiol in TNBC cells but we observed that miR-145-5p expression was induced by hinokitiol. Suppression of miR-145-5p in TNBC cells abolished the inhibitory effect of hinokitiol in mammosphere formation. Overexpression of miR-145-5p in TNBC cells induced LC3-II expression with simultaneously downregulating Akt3 expression. In conclusion, these data suggest that hinokitiol targets BCSCs through the induction of tumor suppressive miRNAs, such as miR-494-3p or miR-145-5p, which could further inhibit oncogenic proteins, such as BMI1 or Akt3. It also suggests that hinokitiol could serve as a potential natural compound for future developing as an anti-breast cancer drug.
URI:	https://ir.csmu.edu.tw:8080/ir/handle/310902500/20877
Appears in Collections:	[生化微生物免疫研究所] 博碩士論文

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