臺灣博碩士論文加值系統

大腸癌是消化道中最常見的惡性腫瘤之一，許多研究證據顯示，癌症幹細胞具有自我更新、導致腫瘤發生和治療抗性的能力，大腸癌類器官的形成與癌幹性、腫瘤發生及抗藥性有關。Abraxane 是一種白蛋白攜帶紫杉醇藥物，已被 FDA 核准用於人類癌症的治療，然而 Abraxane 對人類之癌症類器官中的藥物效果與抗癌機制仍不清楚。在本研究中，我們發現在人類大腸癌 CC020T 類器官中，Abraxane 會經由 p53 與 survivin 相反的角色與表現調控誘導大腸癌 CC020T 類器官的細胞凋亡。Abraxane 誘導 CC020T 癌症類器官的細胞凋亡進而破壞其結構，並同時降低癌幹標誌蛋白的表現，包括 CD133、CD44、Oct4、Nanog 和LGR5。Abraxane 誘發細胞內在路徑中的 caspase 9 活化，進而活化內在路徑的下游 caspase 3 和 PARP 蛋白的分解。再者，Abraxane 增加總 p53 蛋白及磷酸化 p53 中 Ser46 位置的蛋白表現，進而增加 p53 下游蛋白分子 PUMA 和 Bax 的表達而導致細胞凋亡。同時，Abraxane 降低抗凋亡蛋白 survivin 和 Bcl-2 蛋白的表現而誘
導細胞的凋亡。此外，利用 p53 抑制劑 Pifithrin-α 與 Abraxane合併處理 CC020T癌症類器官，會降低 p53 蛋白表現與恢復 CD133 蛋白表現量，相反地，處理survivin 的抑制劑 YM155 與 Abraxane 合併處理 CC020T 癌症類器官，會加強抑制 CD133 的蛋白表現情形。綜合以上結果，我們推測 p53 與 survivin 經由相反的角色調節 Abraxane 所誘導人類大腸癌類器官的細胞凋亡。

Colorectal cancer (CRC) is one of the most common malignant tumors of the digestive system. Studies have demonstrated that cancer stem cells (CSC) are responsible for self-renewing capability, tumor-initiating capacity, and chemoresistance. Cancer organoids are related to cancer stemness, tumorigenesis, and drug-resistance. Abraxane, albumin-bound paclitaxel, has been approved by the FDA for human cancer therapy. However, the drug efficacy and anticancer mechanism of Abraxane in human cancer organoids remain unclear. In this study, we found that p53 and survivin displayed the opposite role in the regulation of Abraxane-induced apoptosis in the human CC020T cancer organoids. Abraxane induced apoptosis by destroying the CC020T cancer organoids structure and the cancer stem cell marker proteins including CD133, CD44, Oct4, Nanog, and LGR5 were reduced. Abraxane elicited the intrinsic pathway of caspase 9 activations. The intrinsic downstream targets of activated-caspase 3 and PARP protein cleavage were induced by Abraxane. Moreover, Abraxane increased total p53 and phosphorylated p53 proteins at Ser46, and further the p53 downstream proteins PUMA and Bax for apoptosis induction. Meantime, Abraxane decreased the levels of anti-apoptotic protein such as survivin and Bcl-2 in the Abraxane-induced apoptosis. Besides, co-treatment with a p53 inhibitor Pifithrin-α and Abraxane reduced the p53 protein levels and restored the CD133 protein levels in the CC020T organoids. In contrast, co-treatment with a survivin inhibitor YM155 and Abraxane decrease the CD133 protein levels in the CC020T organoids. According to the above results, we suggest that p53 and survivin modulate the Abraxane-induced apoptosis through the opposite role in regulating human CRC organoids.

中文摘要 i
Abstract ii
Abbreviations iii
1. Introduction 1
1.1. The stages of colorectal cancer 1
1.2. CRC cancer organoids 1
1.3. The relationship between cancer organoids and cancer stemness proteins 2
1.4. Role of p53 in apoptosis and cancer development 3
1.5. Role of survivin in cancer 4
1.6. The relationship between p53 and survivin in cancer regulation 4
1.7. Abraxane in cancer therapy 5
1.8. The purpose of this study 6
2. Materials and Methods 7
2.1. Chemicals and reagent 7
2.2. Antibodies 7
2.3. 3D tissue culture system of CRC organoids 9
2.4. Western blot analysis 10
2.5. Immunofluorescence staining and confocal microscope 11
2.6. Apoptosis assay using Annexin V-PI stain by flow cytometer analysis 12
2.7. Cell cycle analysis 12
2.8. Preparation of fluorescence nanodiamond-Abraxane 13
2.9. Statistical analysis 13
3. Results and Discussion 14
3.1. Abraxane disrupts organoid structure in the CC020T organoids 14
3.2. Abraxane reduces the cancer stemness related protein levels and destroys the structure of CC020T organoids 15
3.3. Abraxane induces apoptosis by destroying the CC020T cancer organoids structure via reduction of cell adhesion proteins 15
3.4. Abraxane induces apoptosis and mitotic catastrophe in the CC020T organoids 16
3.5. Abraxane induces the intrinsic apoptosis pathway in the CC020T organoids 16
3.6. Abraxane increases the sub-G1 fraction and mitotic catastrophe in the CC020T organoids 17
3.7. Abraxane increases the phosphorylated p53 (Ser46) and its downstream proteins in the CC020T organoids 17
3.8. Abraxane decreases the protein levels of survivin and Bcl-2 in the CC020T organoids 18
3.9. Nanodiamond-labeled Abraxane enters into the CC020T organoids by confocal microscope analysis 19
4. Conclusions 20
5. References 20
6. Figures 27
Fig. 1. Abraxane disrupts the organoid structure in the CC020T organoids 27
Fig. 2. Abraxane reduces the protein levels of cancer stemness and destroys the structure of CC020T organoids. 28
Fig. 3. Abraxane decreases the E-cadherin protein levels and cell-cell adhesion in the CC020T organoids. 29
Fig. 4. Abraxane decreases the β-catenin protein levels in the CC020T organoids 30
Fig. 5. Abraxane decreases the CEACAM5 protein levels in the CC020T organoids. 31
Fig. 6. Abraxane induces apoptosis in CC020T organoids by Annexin V-PI assay under a flow cytometer.. 32
Fig. 7. Abraxane increases apoptosis in CC020T organoids by Annexin V-PI assay under a fluorescence microscope. 33
Fig. 8. Abraxane induces cleaved-forms of caspase 9, caspase 3, and PARP in CC020T organoids. 34
Fig. 9. Abraxane increases the sub-G1 fraction and mitotic arrest by flow cytometer analysis in CC020T organoids.. 35
Fig. 10. Abraxane induces mitotic catastrophe by confocal microscopy analysis in CC020T organoids.. 36
Fig. 11. Abraxane increases the protein levels of total p53 and phospho-p53 (Ser46) in CC020T organoids. 37
Fig. 12. The inhibition of p53 by pifithrin-α restores CD133 protein levels following Abraxane treatment in CC020T organoids.. 38
Fig. 13. The inhibition of survivin by YM155 increases the reduction of CD133 protein levels in CC020T organoids. 39
Fig. 14. Abraxane locates on the surface of the CC020T organoids after 4 h treatment using nanodiamond labeling by confocal microscopy analysis. 40
Fig. 15. Abraxane enters the CC020T organoids after 8 h treatment using nanodiamond labeling by confocal microscopy analysis.. 41
Fig. 16. Cross-sectional scanning of nanodiamond-labeled Abraxane in CC020T organoids. 42
Fig. 17. The model of Abraxane-induced disruption and apoptosis by opposite expression of p53 and survivin. 43
7. Appendixes 44
Appendix 1. The rate of the most common malignancy morbidity and mortality in the world. 44
Appendix 2. The multiple steps and mutations in CRC progression. 45
Appendix 3. The structure of normal organoid and cancer organoid on 3D condition. 46
Appendix 4. Cancer stem cell in cancer therapy. 47

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