臺灣博碩士論文加值系統

肺癌是全球癌症發病率和死亡率的主要原因。雖然小細胞肺癌(Small cell lung cancer; SCLC)僅佔所有肺癌病例的 15%，但其快速生長、早期轉移和抗藥 性導致其致死率高。儘管使用合併 etoposide 及 cisplatin 進行一線化療的反應率 很高，但大多數患者會復發並產生抗藥性。不幸的是，即使使用 topotecan 進行 二線化療也不能有效提高五年生存率。SAHA(Vorinostat)是一種 HDAC 抑製 劑，其具有調控許多抑癌基因轉錄的能力，並在許多癌症細胞株中顯示出抗癌活 性。2-Deoxy-D-glucose(2-DG)是一種糖解作用抑製劑，其可藉由破壞癌細胞的 能量代謝途徑來抑制細胞生長並誘導細胞凋亡。在本研究中，我們藉由檢測細胞 存活率、細胞凋亡、細胞週期分佈和特定訊號傳遞路徑來研究 SAHA 和 2-DG 合 併使用在小細胞肺癌中的抗癌作用。我們的研究表明，與單獨使用 SAHA 或 2- DG 相比，合併使用更能有效地抑制細胞增殖與誘導細胞凋亡。我們還發現，與 單獨使用任何藥物相比，SAHA 與 2-DG 的合併使用導致了更多的 sub-G1 期細 胞週期堆積。此外，我們的研究結果顯示，在 SAHA 單獨使用或與 2-DG 合併使 用時，SAHA 都能夠乙醯化 α-tubulin 和 histone H3。此外，SAHA 和 2-DG 的合 併使用劑量依賴性地降低 S6 的磷酸化。總結來說，這些結果表明 SAHA 和 2- DG 均具有抗癌作用，而合併使用能夠更有效地增加抗癌活性。我們認為此種合 併療法有潛力成為小細胞肺癌患者可靠的治療選擇。

Lung cancer is the leading cause of cancer incidence and mortality worldwide. Although small cell lung cancer (SCLC) accounts for only 15% of all lung cancer cases, its rapid progression, early metastasis and drug resistance lead to high mortality. The response rate to the first-line chemotherapy with the combination of etoposide and cisplatin is high initially, but most patients experience recurrent disease and develop drug resistance. Unfortunately, even the second-line chemotherapy with topotecan cannot effectively improve the five-year survival rate. SAHA (Vorinostat), a HDAC inhibitor, has the ability to regulate the transcription of many tumor suppressor genes and demonstrates anticancer activities in many cancer cell lines. 2-Deoxy-D-glucose (2-DG), a glycolysis inhibitor, inhibits cell growth and induces cell apoptosis by disrupting the energy metabolism of cancer cells. In this study, we investigate the anticancer effects of the combination of SAHA and 2-DG in SCLC by examining cell viability, cell apoptosis, cell cycle distribution and specific signaling pathways. Our data suggested that the combination treatment inhibited cell proliferation and induced cell apoptosis more effectively than either SAHA or 2-DG alone. We also found that SAHA in combination with 2-DG induced more cell cycle accumulation in sub-G1 phase compared with any drug alone. Moreover, our results indicated that SAHA alone or in combination with 2-DG had the ability to acetylate α-tubulin and histone H3. Besides, the combination of SAHA and 2-DG dose-dependently decreased the S6 phosphorylation. In conclusion, these results implied that both SAHA and 2-DG exhibited anticancer effects, while the combination treatment was more effective in these anticancer activities in SCLC cells. We suggest that this combination treatment has the potential to become a promising treatment option for patients with SCLC.

摘要.................................................................................................................................I
Abstract..........................................................................................................................II Contents........................................................................................................................III List of abbreviations.....................................................................................................VI Chapter 1. Introduction..................................................................................................1
1.1 Small cell lung cancer (SCLC) ...............................................................................1
1.2 Treatments for SCLC...............................................................................................1
1.3 Histone deacetylases (HDACs) ...............................................................................2
1.3.1 Functions of HDACs.......................................................................................2 1.3.2 HDAC inhibitors.............................................................................................3 1.3.3 SAHA..............................................................................................................3
1.4 Energy metabolism of cancer cells...........................................................................3 1.4.1 Warburg effect.................................................................................................3 1.4.2 Glycolysis inhibitors.......................................................................................4 1.4.3 2-DG................................................................................................................5
Specific aims..................................................................................................................6 Chapter 2. Materials and Methods.................................................................................7 2.1 Materials...................................................................................................................7
2.1.1 Chemicals........................................................................................................7 2.1.2 Buffers.............................................................................................................8 2.1.3 Reagents..........................................................................................................8 2.1.4 Cell culture media and reagents......................................................................9 2.1.5 Miscellaneous items........................................................................................9 2.1.6 Antibodies.......................................................................................................9
III
2.1.7 Equipment.....................................................................................................10 2.2 Methods..................................................................................................................10 2.2.1 Cell lines and culture.....................................................................................10 2.2.2 Cell viability assay........................................................................................11 2.2.3 Preparation of whole cell lysates...................................................................11 2.2.4 Protein assay..................................................................................................11 2.2.5 SDS-polyacrylamide gel preparation............................................................12
2.2.6 Western blot analysis....................................................................................12 2.2.7 Cell apoptosis analysis..................................................................................13 2.2.8 Cell cycle analysis.........................................................................................14 2.2.9 Hoechst 33342 nuclear staining....................................................................14 2.2.10 Statistical analysis.......................................................................................15
Chapter 3. Results........................................................................................................16 3.1 SAHA reduces cell viability in a dose-dependent manner in SCLC cells.............16 3.2 SAHA in combination with 2-DG effectively reduces cell viability in SCLC cells...............................................................................................................................16 3.3 SAHA in combination with 2-DG enhances apoptosis in SCLC cells..................17 3.4 SAHA in combination with 2-DG promotes apoptosis progression in SCLC cells...............................................................................................................................18 3.5 SAHA in combination with 2-DG induces cell cycle accumulation in sub-G1 phase in SCLC cells.....................................................................................................19 3.6 SAHA in combination with 2-DG induces nuclear morphological changes in SCLC cells...................................................................................................................19 3.7 SAHA in combination with 2-DG regulates SAHA- or 2-DG-related proteins in SCLC cells...................................................................................................................20
IV

Chapter 4. Discussion...................................................................................................22 Reference......................................................................................................................26 Figure 1. The effects of SAHA on cell viability in SCLC cells...................................32 Figure 2. The effects of SAHA, 2-DG, and a combination on cell viability in SCLC cells...............................................................................................................................33 Figure 3. The effects of SAHA, 2-DG, and a combination on protein expression of PARP in SCLC cells....................................................................................................34 Figure 4. Investigation of the effects of SAHA, 2-DG, and a combination on apoptosis progression in SCLC cells by annexin V-FITC and PI staining..................................35 Figure 5. Investigation of the effects of SAHA, 2-DG, and a combination on cell cycle distribution in SCLC cells by PI staining.....................................................................36 Figure 6. Investigation of the effects of SAHA, 2-DG, and a combination on nuclear morphology in SCLC cells by Hoechst 33342 staining...............................................37 Figure 7. The effects of SAHA, 2-DG, and a combination on protein expression of acetyl-α-tubulin, acetyl-histone H3, and p-S6 in SCLC cells......................................38 Appendix......................................................................................................................39 Table 1: Chemicals.......................................................................................................39 Table 2: Buffers............................................................................................................40 Table 3: Reagents.........................................................................................................41 Table 4: Antibodies......................................................................................................41 Table 5: Equipment......................................................................................................42

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