- 學位論文
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
Mechanism Study of Sildenafil as a Chemosensitizer of Doxorubicin against Hormone-Refractory Prostate Cancers
摘要
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。
並列摘要
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.
參考文獻
延伸閱讀
- 林庭卉（2017）。探討Steviol活性衍生物在人類賀爾蒙不依賴型轉移性前列腺癌細胞之抗癌作用機轉〔碩士論文，國立臺灣大學〕。華藝線上圖書館。https://doi.org/10.6342/NTU201703547
- 王韋婷（2012）。第一型組織胺受體拮抗劑在人類雄性激素不依賴型前列腺癌細胞的抗癌作用機轉研究〔碩士論文，國立臺灣大學〕。華藝線上圖書館。https://doi.org/10.6342/NTU.2012.03398
- Wang, C. T. (2017). Ascleposide對人類荷爾蒙不依賴型轉移性前列腺癌之抗癌機轉探討 [master's thesis, National Taiwan University]. Airiti Library. https://doi.org/10.6342/NTU201703192
- 徐瑞苓（2012）。天然物及合成化合物在人類荷爾蒙不依賴型前列腺癌的抗癌機轉探討〔博士論文，國立臺灣大學〕。華藝線上圖書館。https://doi.org/10.6342/NTU.2012.03399
- 黃柏喬（2018）。The study of mechanism underlying sorafenib efficacy in hepatocellular carcinoma cells〔碩士論文，義守大學〕。華藝線上圖書館。https://www.airitilibrary.com/Article/Detail?DocID=U0074-0802201823413000