(3.215.183.251) 您好!臺灣時間:2021/04/23 12:45
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果

詳目顯示:::

我願授權國圖
: 
twitterline
研究生:薛榮宗
研究生(外文):Jung-Tsung Hsueh
論文名稱:研究甘草查爾酮A誘導人類子宮頸癌細胞凋亡和自噬機制
論文名稱(外文):Study on the mechanism of Licochalcone A induces apoptosis and autophagy in human cervical cancer cells
指導教授:謝逸憲
學位類別:碩士
校院名稱:中山醫學大學
系所名稱:生化暨生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2013
畢業學年度:101
語文別:中文
論文頁數:70
相關次數:
  • 被引用被引用:0
  • 點閱點閱:451
  • 評分評分:系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
子宮頸癌是婦女中最常見的癌症之一,與高危險性的人類乳突病毒(HPV, Human Papilloma Virus)感染有關。子宮頸癌造成許多台灣婦女死亡,如果我們能有效地治療子宮頸癌,將可大幅地降低癌症死亡率。類黃酮天然化合物具有多種藥理活性,包括抗氧化、抗發炎、抗癌等活性。甘草查爾酮A是一種類黃酮化合物,已被證實在不同類型的人類癌症細胞具有抗癌特性。然而,目前對於甘草查爾酮A在人類子宮頸癌的抗癌及分子機制,至今仍不清楚。
本實驗以MTT方式證實隨著甘草查爾酮A的濃度和時間增加會抑制人類子宮頸癌SiHa細胞的生長,而且也會降低細胞的群落形成。本研究分兩部分機制探討:(1)我們藉由DAPI和AnnexinV/PI證明甘草查爾酮A調控細胞凋亡伴隨著細胞核濃染、細胞膜PS外翻的現象。此外甘草查爾酮A誘發細胞凋亡會活化caspase-9、-3、PARP和Bcl-2這些蛋白,可是不影響Bax蛋白。(2)當甘草查爾酮A誘發細胞自噬時,我們先觀察自噬作用的指標性蛋白LC3和LC3所聚集形成的粒狀結構,還有透過AVO染色觀察其酸性胞器的形成。此外,甘草查爾酮A透過抑制AKT磷酸化和mTOR磷酸化來啟動其機制功能。而當我們使用3-methyladenine (3MA)抑制自噬作用時造成LC3蛋白表現下降,並且活化caspase相關蛋白表現,因而造成細胞凋亡。我們也有透過實驗證實甘草查爾酮A會誘導ROS的產生。綜合以上結果,說明甘草查爾酮A會誘導人類子宮頸癌細胞自噬和凋亡機制來達到抗癌效果,是未來在人類子宮頸癌上可做為有效的抗癌試劑。


Cervical carcinoma is one of the most frequent cancers in women and is associated with high-risk human papilloma virus (HPV) infection. It causes many women death in Taiwan. If we can treatment to cervical cancer effectively, it will markly decrease the death rate. Natural flavonoids have diverse pharmacological activities, including anti-oxidative, anti-inflammatory, and anti-cancer activities. Licochalcone A is an estrogenic flavonoid that has been shown to have anticancer properties in various types of human cancer cells. However, this study evaluated the anti-cancer effects of Licochalcone A in human cervical cancer cells.
In this study, MTT assay suggested that Licochalcone A inhibits the viability of human cervical carcinoma SiHa cells in a dose- and time-dependent manner and decreased the ability of colony formation. Therefore, this two part of mechanism to study:(1) DAPI stain and Annexin V/PI revealed that Licochalcone A-mediated apoptosis of SiHa cells is accompanied by chromatin condensation and by phosphatidylserine exposure. In addition, Licochalcone A triggered the activations of caspase-9, -3, PARP and Bcl-2 but not affected the protein levels of Bax, resulting in apoptosis induction. (2) The induction of autophagy was detected by monitoring the processing of an autophagy marker LC3, the aggregation of LC3 into granular structures and the formation of acidic organelles by AVO stain. Moreover, Licochalcone A inhibited a sustained activation of the phosphorylation of AKT, and the phosphorylation of mTOR pathways. Inhibition of autophagy using 3-methyladenine (3MA) almost enhanced Licochalcone A inhibited LC3 expression and induced caspases activation and apoptosis in SiHa cells.
Finally, we also found that Licochalcone A induced the reactive oxygen species (ROS) production in SiHa cells. Taken together, we conclude that Licochalcone A exhibits autophagy and apoptosis-mediated antitumor activity in vitro and our results suggest that Licochalcone A might be an effective agent to treat human cervical cancer in the future.

壹. 中文摘要…………………………………………………… 4
貳. 英文摘要…………………………………………………… 5
参. 縮寫表……………………………………………………… 7
肆. 前言………………………………………………………… 8
一. 子宮頸癌…………………………………………………… 8
二. 類黃酮……………………………………………………… 10
三. 甘草查爾酮A……………………………………………… 10
四. 細胞的死亡方式…………………………………………… 12
五. PI3K/AKT/mTOR訊息路徑……………………………… 16
六. 氧化性壓力………………………………………………… 17
伍. 實驗動機…………………………………………………… 19
陸. 實驗材料與方法…………………………………………… 20
一. 實驗材料…………………………………………………… 20
1. 細胞株資料………………………………………………… 20
2. 實驗試劑…………………………………………………… 20
3. 實驗儀器…………………………………………………… 22
二. 實驗方法…………………………………………………… 23
1. 細胞解凍…………………………………………………… 23
2. 人類子宮頸癌細胞繼代培養……………………………… 23
3. 細胞存活率分析…………………………………………… 24
4. 細胞群落實驗……………………………………………… 24
5. DAPI 染色實驗…………………………………………… 25
6. AVO 染色實驗…………………………………………… 25
7. Annexin V/PI雙染實驗…………………………………… 25
8. 全蛋白萃取………………………………………………… 26
9. 蛋白質定量測定…………………………………………… 26
10. 西方墨點法………………………………………………… 27
11. 免疫螢光法………………………………………………… 28
12. 抽取中量質體……………………………………………… 28
13. 轉染作用…………………………………………………… 29
14. 自由基測定………………………………………………… 30
15. 統計分析…………………………………………………… 30
柒. 實驗結果…………………………………………………… 31
一. 甘草查爾酮 A對於人類子宮頸癌細胞株存活率的影響……………………………………………………………
31
二. 甘草查爾酮A對於人類子宮頸癌細胞群落形成的影響……………………………………………………………
31
三. 甘草查爾酮 A誘發人類子宮頸癌SiHa細胞有細胞凋亡之現象………………………………………………………
32
四. 甘草查爾酮 A 對caspase蛋白、Bcl-2和Bax的影響………………………………………………..…………..
32
五. 甘草查爾酮 A誘發人類子宮頸癌SiHa細胞有細胞自噬之現象…………….…………………………………………
33
六. 甘草查爾酮 A對於細胞自噬相關蛋白的影響…………... 34
七. 抑制細胞自噬探討甘草查爾酮A對SiHa細胞之影響….. 35
八. 甘草查爾酮A誘導人類子宮頸癌細胞在GFP-LC3之表現………………………………..…………………………..
36
九. 甘草查爾酮A誘發人類子宮頸癌SiHa細胞有自噬現象是否透過PI3K/AKT/mTOR訊息路徑………………………
37
十. 甘草查爾酮A誘發人類子宮頸癌SiHa細胞有自噬現象是否受到氧化性壓力………………………………………….
38
捌. 討論………………………………………………………… 39
玖. 參考文獻…………………………………………………… 43
拾. 圖表與圖表說明…………………………………………… 47
拾壹. 附錄………………………………………………………… 67


1.Vaccarella, S., et al., Worldwide trends in cervical cancer incidence: Impact of screening against changes in disease risk factors. Eur J Cancer, 2013.
2.Snijders, P.J., et al., HPV-mediated cervical carcinogenesis: concepts and clinical implications. J Pathol, 2006. 208(2): p. 152-64.
3.da Rocha, A.B., R.M. Lopes, and G. Schwartsmann, Natural products in anticancer therapy. Curr Opin Pharmacol, 2001. 1(4): p. 364-9.
4.Havsteen, B.H., The biochemistry and medical significance of the flavonoids. Pharmacol Ther, 2002. 96(2-3): p. 67-202.
5.Nicolini, F., et al., Induction of G /M phase arrest and apoptosis by the flavonoid tamarixetin on human leukemia cells. Mol Carcinog, 2013.
6.Jiang, D., D. Li, and W. Wu, Inhibitory effects and mechanisms of luteolin on proliferation and migration of vascular smooth muscle cells. Nutrients, 2013. 5(5): p. 1648-59.
7.Feng, S., et al., Polyprenylated isoflavanone and isoflavonoids from Ormosia henryi and their cytotoxicity and anti-oxidation activity. Fitoterapia, 2012. 83(1): p. 161-5.
8.Xia, J.F., et al., Flavonoids as potential anti-hepatocellular carcinoma agents: Recent approaches using HepG2 cell line. Drug Discov Ther, 2013. 7(1): p. 1-8.
9.Ren, W., et al., Flavonoids: promising anticancer agents. Med Res Rev, 2003. 23(4): p. 519-34.
10.Zhou, Y., et al., HQS-3, a newly synthesized flavonoid, possesses potent anti-tumor effect in vivo and in vitro. Eur J Pharm Sci, 2013.
11.Hassan, S.W., et al., Phytochemical and toxicological studies of aqueous leaves extracts of Erythrophleum africanum. Pak J Biol Sci, 2007. 10(21): p. 3815-21.
12.Messier, C., et al., Licorice and its potential beneficial effects in common oro-dental diseases. Oral Dis, 2012. 18(1): p. 32-9.
13.Shibata, S., A drug over the millennia: pharmacognosy, chemistry, and pharmacology of licorice. Yakugaku Zasshi, 2000. 120(10): p. 849-62.
14.Chu, X., et al., Licochalcone a inhibits lipopolysaccharide-induced inflammatory response in vitro and in vivo. J Agric Food Chem, 2012. 60(15): p. 3947-54.
15.Kim, Y.H., et al., Antiangiogenic effect of licochalcone A. Biochem Pharmacol, 2010. 80(8): p. 1152-9.
16.Xiao, X.Y., et al., Licochalcone A inhibits growth of gastric cancer cells by arresting cell cycle progression and inducing apoptosis. Cancer Lett, 2011. 302(1): p. 69-75.
17.Lee, C.S., et al., Guanylate cyclase activator YC-1 potentiates apoptotic effect of licochalcone A on human epithelial ovarian carcinoma cells via activation of death receptor and mitochondrial pathways. Eur J Pharmacol, 2012. 683(1-3): p. 54-62.
18.Park, J.H., et al., Anti-proliferative effect of licochalcone A on vascular smooth muscle cells. Biol Pharm Bull, 2008. 31(11): p. 1996-2000.
19.Kim, J.K., et al., Antitumor and antimetastatic effects of licochalcone A in mouse models. J Mol Med (Berl), 2010. 88(8): p. 829-38.
20.Yo, Y.T., et al., Licorice and licochalcone-A induce autophagy in LNCaP prostate cancer cells by suppression of Bcl-2 expression and the mTOR pathway. J Agric Food Chem, 2009. 57(18): p. 8266-73.
21.Diamantis, A., et al., A brief history of apoptosis: from ancient to modern times. Onkologie, 2008. 31(12): p. 702-6.
22.Kerr, J.F., A.H. Wyllie, and A.R. Currie, Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer, 1972. 26(4): p. 239-57.
23.Fadeel, B. and S. Orrenius, Apoptosis: a basic biological phenomenon with wide-ranging implications in human disease. J Intern Med, 2005. 258(6): p. 479-517.
24.Danial, N.N. and S.J. Korsmeyer, Cell death: critical control points. Cell, 2004. 116(2): p. 205-19.
25.Earnshaw, W.C., L.M. Martins, and S.H. Kaufmann, Mammalian caspases: structure, activation, substrates, and functions during apoptosis. Annu Rev Biochem, 1999. 68: p. 383-424.
26.Ravikumar, B., et al., Regulation of mammalian autophagy in physiology and pathophysiology. Physiol Rev, 2010. 90(4): p. 1383-435.
27.Mizushima, N. and M. Komatsu, Autophagy: renovation of cells and tissues. Cell, 2011. 147(4): p. 728-41.
28.Rosenfeldt, M.T. and K.M. Ryan, The role of autophagy in tumour development and cancer therapy. Expert Rev Mol Med, 2009. 11: p. e36.
29.Klionsky, D.J., The molecular machinery of autophagy: unanswered questions. J Cell Sci, 2005. 118(Pt 1): p. 7-18.
30.Chen, Y. and D.J. Klionsky, The regulation of autophagy - unanswered questions. J Cell Sci, 2011. 124(Pt 2): p. 161-70.
31.Das, G., B.V. Shravage, and E.H. Baehrecke, Regulation and function of autophagy during cell survival and cell death. Cold Spring Harb Perspect Biol, 2012. 4(6).
32.Debnath, J., E.H. Baehrecke, and G. Kroemer, Does autophagy contribute to cell death? Autophagy, 2005. 1(2): p. 66-74.
33.Burris, H.A., 3rd, Overcoming acquired resistance to anticancer therapy: focus on the PI3K/AKT/mTOR pathway. Cancer Chemother Pharmacol, 2013. 71(4): p. 829-42.
34.Owonikoko, T.K. and F.R. Khuri, Targeting the PI3K/AKT/mTOR Pathway. Am Soc Clin Oncol Educ Book, 2013. 2013: p. 395-401.
35.Scherz-Shouval, R. and Z. Elazar, ROS, mitochondria and the regulation of autophagy. Trends Cell Biol, 2007. 17(9): p. 422-7.
36.Scherz-Shouval, R. and Z. Elazar, Regulation of autophagy by ROS: physiology and pathology. Trends Biochem Sci, 2011. 36(1): p. 30-8.
37.Azad, M.B., Y. Chen, and S.B. Gibson, Regulation of autophagy by reactive oxygen species (ROS): implications for cancer progression and treatment. Antioxid Redox Signal, 2009. 11(4): p. 777-90.
38.Moon, Y.J., X. Wang, and M.E. Morris, Dietary flavonoids: effects on xenobiotic and carcinogen metabolism. Toxicol In Vitro, 2006. 20(2): p. 187-210.
39.Xu, H.D., et al., The pro-survival role of autophagy depends on Bcl-2 under nutrition stress conditions. PLoS One, 2013. 8(5): p. e63232.
40.Yin, X., N. Zhang, and W. Di, Regulation of LC3-dependent protective autophagy in ovarian cancer cells by protein phosphatase 2A. Int J Gynecol Cancer, 2013. 23(4): p. 630-41.
41.Gao, P., et al., The Bcl-2 homology domain 3 mimetic gossypol induces both Beclin 1-dependent and Beclin 1-independent cytoprotective autophagy in cancer cells. J Biol Chem, 2010. 285(33): p. 25570-81.
42.Yun, S.M., et al., Tanshinone IIA Induces Autophagic Cell Death via Activation of AMPK and ERK and Inhibition of mTOR and p70 S6K in KBM-5 Leukemia Cells. Phytother Res, 2013.
43.Dando, I., et al., Cannabinoids inhibit energetic metabolism and induce AMPK-dependent autophagy in pancreatic cancer cells. Cell Death Dis, 2013. 4: p. e664.
44.Shin, S., et al., The Omega-3 Polyunsaturated Fatty Acid DHA Induces Simultaneous Apoptosis and Autophagy via Mitochondrial ROS-Mediated Akt-mTOR Signaling in Prostate Cancer Cells Expressing Mutant p53. Biomed Res Int, 2013. 2013: p. 568671.
45.Xu, Y., et al., Inhibition of autophagy enhances cisplatin cytotoxicity through endoplasmic reticulum stress in human cervical cancer cells. Cancer Lett, 2012. 314(2): p. 232-43.
46.Dalton, L.E., et al., The endoplasmic reticulum stress marker CHOP predicts survival in malignant mesothelioma. Br J Cancer, 2013. 108(6): p. 1340-7.
47.Mathew, R., V. Karantza-Wadsworth, and E. White, Role of autophagy in cancer. Nat Rev Cancer, 2007. 7(12): p. 961-7.
48.Lin, J.F., et al., Benzyl isothiocyanate induces protective autophagy in human prostate cancer cells via inhibition of mTOR signaling. Carcinogenesis, 2013. 34(2): p. 406-14.
49.Verschooten, L., et al., Autophagy inhibitor chloroquine enhanced the cell death inducing effect of the flavonoid luteolin in metastatic squamous cell carcinoma cells. PLoS One, 2012. 7(10): p. e48264.
50.Chen, S., et al., Autophagy is a therapeutic target in anticancer drug resistance. Biochim Biophys Acta, 2010. 1806(2): p. 220-9.
51.Aredia, F. and A.I. Scovassi, Manipulation of autophagy in cancer cells: an innovative strategy to fight drug resistance. Future Med Chem, 2013. 5(9): p. 1009-21.
52.Chen, K.L., et al., Targeting cathepsin S induces tumor cell autophagy via the EGFR-ERK signaling pathway. Cancer Lett, 2012. 317(1): p. 89-98.


QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top
系統版面圖檔 系統版面圖檔