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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 顧記華 | |
dc.contributor.author | Jo-Fan Chang | en |
dc.contributor.author | 張若凡 | zh_TW |
dc.date.accessioned | 2021-05-19T17:56:39Z | - |
dc.date.available | 2021-08-26 | |
dc.date.available | 2021-05-19T17:56:39Z | - |
dc.date.copyright | 2016-08-26 | |
dc.date.issued | 2016 | |
dc.date.submitted | 2016-08-18 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/7869 | - |
dc.description.abstract | 近年來,老藥新用在藥物開發上的發展潛力逐漸被受重視,像是威而鋼其學名sildenafil (Phosphodiesterase-5抑制劑),為治療男性勃起功能障礙的藥物,但在最近研究發現,其在活體內外實驗中都具有加強doxorubicin毒殺賀爾蒙不依賴型前列腺癌細胞的能力,但其相關分子機制仍不清楚。在我們的研究結果中發現,sildenafil本身並不會影響賀爾蒙不依賴型前列腺癌細胞 (PC-3及DU145) 的存活率,但是當其與doxorubicin同時作用下,sildenafil能協同性地增強doxorubicin所誘導的前列腺癌細胞之細胞凋亡現象,包括增加核小體DNA斷裂、sub-G1細胞數量、內因性與外因性細胞凋亡訊號,並減少抗凋亡Bcl-2蛋白質家族的表現量 (Mcl-1, Bcl-xL及Bcl-2)。此外,值得注意的是,雖然合併使用sildenafil與doxorubicin能增加PC-3細胞中的氧化壓力,但當我們利用抗氧化劑 (NAC及trolox) 來去除該氧化壓力後,卻無法阻止細胞凋亡的發生,意味著氧化壓力並非造成藥物合併後細胞凋亡增加之原因。有趣的是,我們也發現sildenafil可以加劇其他第二型拓譜異構酶 (etoposide及mitoxantrone) 所產生之細胞凋亡情況,然而 sildenafil對於第一型拓譜異構酶 (camptothecin) 所導致的細胞凋亡則沒有影響。已知doxorubicin毒殺癌細胞的其一機制是增加DNA雙骨斷裂的產生,我們進一步探討sildenafil對於doxorubicin所誘導的DNA損傷及其修補機制之影響。實驗結果發現,doxorubicin造成的DNA雙骨斷裂之兩種修復機制 (homologous recombination, HR; non-homologous DNA end joining, NHEJ) 都會被sildenafil所抑制,例如:合併使用sildenafil與doxorubicin會減少HR修補機制中RPA32的過量磷酸化、降低Rad51之表現量及其在細胞核內形成foci的能力;並在NHEJ修補機制中,減少DNA-PKcs (Thr2609)的磷酸化、抑制Ku80鍵結到DNA斷裂尾端之能力。另外,已知sildenafil為phosphodiesterase-5 (PDE5) 之抑制劑,我們也進一步探討抑制PDE5對於sildenafil增強doxorubicin毒殺效果的重要性。實驗結果顯示,不論是利用其他PDE5活性抑制劑 (vardenafil或tadalafil),或是使用siRNA 去抑制PDE5的表現,都可以加強doxorubicin之細胞毒殺效果,然而我們卻發現只有PDE5活性抑制劑才能有效減少doxorubicin所誘導的DNA雙骨斷裂HR修補機制。總結來說,sildenafil可經由抑制HR與NHEJ途徑來減少doxorubicin所誘導的DNA雙骨斷裂之修補,導致核小體DNA片段化與內外因性細胞凋亡信號增強,藉此增加doxorubicin毒殺賀爾蒙不依賴型前列腺癌細胞之能力。而目前我們不排除PDE5在此機制中可能扮演部分腳色,但這仍須要更進一步的研究去驗證。 | zh_TW |
dc.description.abstract | Drug repositioning is a potential strategy for drug development. Recently, sildenafil, a phosphodiesterase-5 (PDE5) inhibitor, has been repurposed as a chemosensitizer to synergistically potentiate doxorubicin-induced cell killing in hormone-refractory prostate cancer (HRPC) both in vitro and in vivo. However the synergistic anticancer mechanism has not been well identified. In the present study, the data demonstrated that sildenafil by itself did not affect cell survival of PC-3 and DU145 (two HRPC cell lines), but significantly enhanced cell apoptosis induced by doxorubicin, as evidenced by the synergistic increase of nucleosomal DNA fragments and sub-G1 (apoptosis) population, and the activation of both intrinsic and extrinsic apoptotic pathways. Moreover, the anti-apoptotic Bcl-2 family proteins, Mcl-1, Bcl-xL and Bcl-2, were significantly downregulated by the combinatorial treatment with sildenafil and doxorubicin. It is noteworthy that an increase in cellular ROS at early combinatorial treatment was noted; however, both ROS scavengers, NAC and trolox, dramatically abolished the ROS production but failed to inhibit cell apoptosis, indicating the sensitization mechanism beyond the oxidative stress. Interestingly, sildenafil also enhanced cell apoptosis induced by other topoisomerase II inhibitors (e.g., etoposide and mitoxantrone) but not topoisomerase I inhibitor (e.g., camptothecin). Due to DNA-damaging properties of doxorubicin, the regulators and signaling of DNA double-strand break (DSB) and repair pathways were studied. As a result, the combinatorial treatment reduced the protein expression of hyperphosphorylated RPA32 and phosphorylated DNA-PKcs (Thr2609), which were involved in DSB repair pathways, homologous recombination (HR) and non-homologous DNA end joining (NHEJ), respectively. The defects in HR and NHEJ pathways were further substantiated by the reduced levels of nuclear Rad51 foci formation and DNA end-binding activity of nuclear Ku80. The role of PDE5 in the sensitization mechanism was examined as well. The data revealed that inhibition of PDE5 activity by two other inhibitors, vardenafil or tadalafil, or PDE5 knockdown by siRNA potentiated the cell-killing effect of doxorubicin. However, only PDE5 inhibitors but not PDE5 knockdown reduced the HR-mediated DSB repair in response to doxorubicin. In conclusion, the data suggest that sildenafil enhances doxorubicin-induced apoptosis in HRPC through the impairment of HR and NHEJ-mediated DSB repair systems, leading to synergistic increase of nucleosomal DNA fragments and activation of both intrinsic and extrinsic apoptotic pathways. Inhibition of PDE5 is, at least partly, responsible for the sensitization mechanism. | en |
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dc.description.tableofcontents | 口試委員會審定書 ......................................i
致謝..................................................ii List of Abbreviations................................iii 中文摘要...............................................v Abstract.............................................vii Contents..............................................ix Aim of the study.......................................1 Chapter 1: Introduction................................3 1.1. Prostate..........................................3 1.2. Prostate cancer...................................3 1.3. Human Prostate cancer cell lines..................6 1.4. Doxorubicin.......................................7 1.5. Sildenafil........................................7 1.6. Cell death.......................................10 1.7. Apoptosis........................................11 1.8. Oxidative stress.................................14 1.9. DNA double-strand break signaling and repair.....15 Chapter 2: Materials and Methods......................19 Chapter 3: Results....................................26 3.1. Effect of doxorubicin and sildenafil on cell cycle progression in PC-3 and DU145 cells...................26 3.2. Validation of sildenafil-mediated sensitization of doxorubicin-induced apoptosis.........................26 3.3. Effect of doxorubicin or/and sildenafil on the expression of Bcl-2 family proteins...................27 3.4. Effect of doxorubicin or/and sildenafil on ROS production............................................28 3.5. Effect of sildenafil on doxorubicin-induced DNA double-strand break signaling and repair system.......28 3.6. Effect of combinatorial treatment on DNA end-binding capacity and protein expression of Ku80...............30 3.7. Effect of combinatorial treatment on nuclear foci formation and expression of Rad51.....................30 3.8. Effect of other PDE5 inhibitors on doxorubicin-induced cell apoptosis................................31 3.9. Effect of other PDE5 inhibitors on HR-mediated repair of doxorubicin-induced DSB.....................31 3.10. Effect of PDE5 knockdown on doxorubicin-induced cell death and DSB signaling and repair...............32 3.11. Effect of sildenafil on the sensitization of apoptosis induced by other topoisomerase inhibitors...33 Chapter 4: Discussions................................34 4.1. Effect of sildenafil on apoptosis induced by doxorubicin in PC-3 cells.............................34 4.2. The role of ROS production in the sensitization mechanism.............................................35 4.3. Effect of sildenafil on doxorubicin-induced DNA double-strand break signaling and repair..............36 4.3.1. DNA double-strand break signaling..............37 4.3.2. DNA double-strand break repair.................38 4.4. Effect of PDE5 inhibitors or PDE5 knockdown on doxorubicin-induced cell death and DSB signaling and repair................................................41 4.5. Effect of other topoisomerase inhibitors or/and sildenafil on cell apoptosis..........................42 Chapter 5: Conclusion.................................44 Tables Table 1. The stages and estimated 5-year survival rates for prostate cancers...................................5 Table 2. The most commonly used human prostate cancer cell lines for research................................6 Table 3. PDE5 inhibitors approved by FDA...............8 Table 4. Proposed mechanisms for sildenafil as a chemosensitizer in cancers.............................9 Figures Figure 1. Effect of doxorubicin and/or sildenafil on cell cycle distribution in PC-3 cells......................46 Figure 2. Effect of doxorubicin and/or sildenafil on cell cycle distribution in DU145 cells.....................47 Figure 3. Effect of sildenafil on doxorubicin-induced apoptosis in PC-3 cells...............................49 Figure 4. Effect of doxorubicin and/or sildenafil on the expression of Bcl-2 family in PC-3 cells..............51 Figure 5. Effect of doxorubicin and/or sildenafil on ROS production in PC-3 cells..............................52 Figure 6. Effect of ROS scavengers NAC and trolox on cell apoptosis induced by doxorubicin and sildenafil.......53 Figure 7. Effect of doxorubicin and/or sildenafil on the integrity of chromosomal DNA in PC-3 cells............54 Figure 8. Effect of sildenafil on doxorubicin-induced DNA double-strand break (DSB) signaling and repair in PC-3 cells.................................................57 Figure 9. Effect of sildenafil on doxorubicin-induced DNA double-strand break (DSB) signaling and repair in DU145 cells.................................................58 Figure 10. Effect of doxorubicin and/or sildenafil on DNA end-binding capacity, total and nuclear expression of Ku80 in PC-3 cells....................................59 Figure 11. Effect of doxorubicin and/or sildenafil on the nuclear foci formation and expression of Rad51 in PC-3 cells.................................................61 Figure 12. Effect of other PDE5 inhibitors, vardenafil and tadalafil, on doxorubicin-induced cell apoptosis in PC-3cells.............................................63 Figure 13. Effect of other PDE5 inhibitors, vardenafil and tadalafil, on doxorubicin-induced cell apoptosis in DU145 cells...........................................64 Figure 14. Effect of PDE5 inhibitors, vardenafil and tadalafil, on HR-mediated repair of DSB induced by doxorubicin in PC-3 cells.............................65 Figure 15. Correlation between Rad51 expression and the level of nucleosomal DNA fragments (apoptosis) in PC-3 cells treated with doxorubicin or in combination with different PDE5 inhibitors.............................66 Figure 16. Effect of PDE5 knockdown on doxorubicin-induced cell death in PC-3cells.......................67 Figure 17. Effect of PDE5 knockdown on doxorubicin-induced DSB signalling and repair in PC-3 cells.......68 Figure 18. Effect of other topoisomerase inhibitors and/or sildenafil on cell death in PC-3 cells.........70 Figure 19. A schematic of how sildenafil sensitizes HRPC cells to chemotherapy drug doxorubicin................71 Appendixes............................................72 References............................................76 | |
dc.language.iso | en | |
dc.title | 探討Sildenafil增強化療藥物Doxorubicin在人類荷爾蒙不依賴型前列腺癌細胞之抗癌作用機轉 | zh_TW |
dc.title | Mechanism Study of Sildenafil as a Chemosensitizer of Doxorubicin against Hormone-Refractory Prostate Cancers | en |
dc.type | Thesis | |
dc.date.schoolyear | 104-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 許麗卿,黃聰龍,蕭哲志 | |
dc.subject.keyword | Doxorubicin,Sildenafil,Hormone-refractory prostate cancer,HR,NHEJ,PDE5,Apoptosis, | zh_TW |
dc.relation.page | 83 | |
dc.identifier.doi | 10.6342/NTU201602842 | |
dc.rights.note | 同意授權(全球公開) | |
dc.date.accepted | 2016-08-18 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 藥學研究所 | zh_TW |
顯示於系所單位: | 藥學系 |
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