研究目的：誘導自噬性細胞死亡已被證明是一種治療固態惡性腫瘤的有效方法，目前利用這項途徑來治療泌尿上皮癌的經驗仍缺乏，泌尿上皮癌是一種常見的男性腫瘤，尤其常見於膀胱癌，一旦發生轉移或化學治療抗藥性，往往無藥可醫。在這項研究中，我們使用兩個藥物來研究克服化學治療抗藥性；一個是新型的雙效抑制劑NVP-BEZ235，它是phosphatidylinositol 3-kinase（PI3K）和mammalian target of rapamycin（mTOR）抑制劑，及靈芝中提煉出的免疫調節蛋白ganoderma tsugae (FIP-gts) ，這兩個藥物都具有抗實體腫瘤的抗癌活性，從治療泌尿上皮癌（UC）的細胞株實驗中，了解其中的抗癌機轉。
研究材料和方法：兩個UC細胞株，NTUB1以及cisplatin抗藥性的N / P（14）是我們主要的實驗對象，我們投以NVP-BEZ235及FIP-gts，使用細胞存活率分析（MTT assay）及流式細胞儀來觀察細胞的毒殺作用，也利用酸性囊狀胞器（Acidic vesicular organells, AVO）的染色與西方墨點法（western blotting）來了解及驗證細胞死亡的機制。
研究結果：我們發現兩種截然不同的細胞自噬作用及結果；NVP-BEZ235可以有效抑制原生及cisplatin抗藥性泌尿上皮癌細胞株的生長，它透過降低p62蛋白和磷酸化Rb的表達，造成GFP-LC3的轉換及產生GFP的裂解，而引發細胞自噬作用及細胞死亡，我們從酸性囊泡胞器的產生，驗證了這項發現，同時，加入細胞自噬抑制劑chloroquine (CQ)，會反向提高細胞存活，因此，CQ抑制細胞自噬波(autophagy flux)似乎反而阻斷了NVP-BEZ235的抗癌效果；另一方面，FIP-gts透過抑制mTOR的路徑，也具有促進細胞自噬及毒殺泌尿上皮癌細胞的功能，但若加入細胞自噬的抑制劑，bafilomycin-A1（Baf-A1）或是chloroquine(CQ)，都可以有效阻擾細胞自噬作用的進行，但是反而會提高細胞凋亡(apoptosis)作用，造成更大量的泌尿上皮癌細胞死亡。
結論：我們的研究發現了兩種抑制泌尿上皮癌細胞增殖，尤其是cisplatin抗藥性細胞的模式，這兩種模式都與細胞自噬作用有關，其一是如NVP-BEZ235直接產生大量細胞自噬，並且細胞死亡，其間若加入細胞自噬抑制劑，反而會造成細胞存活；其二是在誘導細胞自噬時，同時加入細胞自噬抑制劑，但反而誘導造成凋亡性細胞死亡。我們的研究結果表明，NVP-BEZ235的抗癌功效是透過提高細胞自噬通量，若加入CQ，會抵消了毒殺癌細胞的效果。此外，FIP-gts的實驗中發現，FIP-gts加入Baf-A1或CQ，會促進細胞凋亡作用。因此，透過誘導細胞自噬及細胞凋亡的相互轉換，將成為治療抗化藥性泌尿上皮癌的治療方向。

Purpose: Therapeutically induced autophagic cell death has been proven to be effective in cases of solid tumors. The dual phosphatidylinositol 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) inhibitor NVP-BEZ235 possesses antitumor activity against solid tumors. Inhibition of mTOR has been shown to elicit autophagy. We examined the antiproliferation and autophagic activities of NVP-BEZ235 in parental and cisplatin-resistant urothelial carcinoma (UC) cells. Besides, we tested a fungal immunomodulatory protein, ganoderma tsugae (FIP-gts) which possesses antitumor activity against solid tumors and inhibits telomerase activity. FIP-gts induces autophagy in cancer cells and may provide an alternative pathway against chemo-resistance. Materials and Methods: Two UC cell lines, the parental NTUB1 cell line and cisplatin resistant N/P(14) cell line were used to investigate the cytotoxicity effects and the autophagy regulation of NVP-BEZ235 and FIP-gts. MTT assay and flow cytometry were used for cell viability change and cell death during treatment. Acidic vesicular organelles (AVO) staining was used for the detection of autophagosome formation. Western blotting was used for the validation of the protein level change during cell death. Results: NVP-BEZ235 was effective in inhibiting the growth of UC cells including parental and cisplatin resistant cells. It elicited acidic vesicular organelle (AVO) development on flow cytometry and induced the decrease of p62 and phospho-Rb expressions in a concentration-dependent manner. GFP-LC3 conversion and the appearance of cleaved-GFP following NVP-BEZ235 treatment were demonstrated on Western blot. In addition, lysosomotropic inhibition of autophagy by chloroquine (CQ), resulted in proliferation of UC cells. Thus, inhibition of autophagic flux by CQ counteracts the anticancer effects of NVP-BEZ235. FIP-gts and bafilomycin-A1 (Baf-A1) and or chloroquine (CQ) could enhance a significantly synergistic cytotoxicity. FIP-gts itself showed a promising autophagy effect in the promoting cancer cell death. While adding on autophagy inhibitor Baf-A1 or CQ, the final effect leading to an increase of apoptosis. Conclusion: NVP-BEZ235 induced autophagy can overcome the chemo-resistant UC cell lines and can be an alternative treatment pathway. Inhibition of the NVP-BEZ235 induced autophagy may lead to a cancer cell recovery. In the contrary, blockade of FIP-gts induced autophagy pathway may lead to significant cancer cell apoptosis. Application of cross talk between apoptosis and autophagy, can be another treatment route against chemo-resistance.

1.中文摘要................................................1
2.Abstract................................................3
3.Abbreviations...........................................6
4.Introduction............................................7
4.1General concept of urothelial carcinoma................7
4.2Epidemiology...........................................9
4.3Genetic expression and predisposing factors...........10
4.4Occupational carcinogen...............................11
4.5Other risk factors of bladder cancer..................11
4.6Bladder cancer pathology..............................13
4.7Clinical staging of bladder cancer....................14
4.8Molecular biology expression of urothelial carcinoma..15
4.9Surgical intervention of urothelial carcinoma.........16
4.10Chemotherapy of urothelial carcinoma.................17
4.11Difficulty in the treatment of urothelial carcinoma..17
4.12Current new model against chemoresistance in urothelial carcinoma................................................18
4.13Autophagy is a new field against chemoresistance.....19
4.14NVP-BEZ235...........................................21
4.15Ganoderma tsugae (FIP-gts)...........................22
5.Motivation.............................................24
6.Materials and Methods..................................25
6.1NVP-BEZ235 studies....................................25
6.1.1 Cell line and chemicals.........................25
6.1.2 Cell viability assay............................25
6.1.3 Western blot....................................26
6.1.4 Assessment of apoptotic cell death..............28
6.1.5 Analysis of cell cycle distribution.............28
6.1.6 Detection & quantification of acidic vascular organelle development....................................29
6.1.7 Assessment of autophagic cell death.............29
6.2 FIP-gts studies.................................30
6.2.1 Cell line and chemicals.........................30
6.2.2 Cell viability assay............................31
6.2.3 Assessment of apoptotic cell death..............31
6.2.4 Detection & quantification of acidic vascular organelle development....................................32
6.2.5 Western blot....................................32
7.Results................................................34
7.1 NVP-BEZ235 reduces cell viability but does not significant induce apoptosis in urothelical carcinoma cells....................................................34
7.2 NVP-BEZ235 induces G1 cell cycle arrest in NTUB1 and N/P(14) cells........................................36
7.3 Effect of NVP-BEZ235 on the phosphorylation of Akt/mTOR signaling pathway in urothelial carcinoma cells....................................................38
7.4 NVP-BEZ235 induces autophagy in NTUB1 and N/P(14) cells....................................................39
7.5 The effect of 3-MA and chloroquine combine NVP-BEZ235 treatment in NTUB1 and N/P(14) cells..............40
7.6 FIP-gts combine treatment with bafilomycin-A1 and chloroquine accelerates cell death in NTUB1 and N/P cells....................................................43
7.7 FIP-gts induces autophagy in urothelial carcinoma cells....................................................45
7.8 The effect of FIP-gts combined Baf-A1 treatment in NTUB1 and N/P cells...................................46
7.9 Downregulation of Beclin-1 expression is not associated with ubiquitin-proteasome pathway.............48
8.Discussion.............................................50
9.Conclusions............................................60
10.References............................................61
11.Figures and Tables....................................71
Figure 1. Cytotoxic effects of cisplatin, NVP-BEZ235, LY294002 and rapamycin in NTUB1, N/P(14) cells...........71
Figure 2. NVP-BEZ235 does not significantly induce apoptosis in NTUB1 and N/P(14) cells.....................72
Figure 3. NVP-BEZ235 induces G1 cell cycle arrest in NTUB1 and N/P(14) cells........................................73
Figure 4. Effect of NVP-BEZ235 on protein expressions of cell-cycle regulators....................................74
Figure 5. Effect of NVP-BEZ235 on the phosphorylation of Akt/mTOR signaling pathways..............................75
Figure 6. Induction of autophagy in urothelial cells by NVP-BEZ235 treatment.........................................76
Figure 7. Cytotoxic effects of chloroquine and 3-MA combine treatment with NVP-BEZ235 in NTUB1, N/P(14) cells........77
Figure 8. Effect of NVP-BEZ235 combine treatment with chloroquine on cell cycle distribution and protein expressions in NTUB1 and N/P(14) cells...................78
Figure 9. Effect of NVP-BEZ235 combine treatment with chloroquine on protein expressions in NTUB1 and N/P(14) cells....................................................79
Figure 10. Cytotoxic effects of 3-methyladenine (3-MA), bafilomycin-A1 (Baf-A1) and chloroquine (CQ) combine treatment with gts in NTUB1, N/P cells...................80
Figure 11. The detection of apoptosis in NTUB1 and N/P cells exposed to gts and Baf-A1..........................81
Figure 12.The detection of autophagy in NTUB1 and N/P cells exposed to gts and Baf-A1................................82
Figure 13. Effect of FIP-gts combine treatment with Baf-A1 on protein expressions in NTUB1 cells....................83
Figure 14. Effect of FIP-gts combine treatment with Baf-A1 on protein expressions in N/P cells......................84
Figure 15. Effect of FIP-gts and Baf-A1 on degradation of Beclin-1.................................................85
12.Appendix..............................................86
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