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研究生:周柔君
研究生(外文):Jou-Chun Chou
論文名稱:辣椒素對皮膚癌 A375 細胞株中 tNOX 及細胞存活的調控
論文名稱(外文):The effects of capsaicin on tNOX expression and cell survival in A375 cells.
指導教授:葛其梅葛其梅引用關係
指導教授(外文):Chi-Mei Hsueh
口試委員:蕭乃文
口試日期:2012-12-28
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生命科學系所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2012
畢業學年度:101
語文別:中文
論文頁數:50
中文關鍵詞:細胞自噬
外文關鍵詞:autophagy
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Tumor-associated NADH oxidase (tNOX)是與腫瘤細胞生長相關的蛋白,存在於轉型細胞中。研究發現高濃度的辣椒素處理會抑制 tNOX 的活性與蛋白表現量,並抑制細胞的生長和導致細胞凋亡。然而在 HCT116 細胞株中,在辣椒素濃度 10 μM 以下處理,卻會增加 tNOX 蛋白表現量、細胞生長和移動能力,也會誘發上皮間質轉化現象,顯示 tNOX 蛋白表現量與細胞癌化有關。在此篇研究中,我們利用人類黑色素癌細胞株為實驗模式,探討不同濃度的辣椒素對 A375 細胞株的影響。結果發現以濃度高於 50 μM 辣椒素處理,A375 細胞生長受到抑制,而在 400 μM 濃度時,會造成細胞凋亡,也伴隨著 tNOX 蛋白表現量減少;但低濃度 10 μM 處理下,在 72 小時的時候會增進細胞生長。由於在許多腫瘤細胞發生細胞自噬可幫助細胞增加存活或逃避細胞凋亡,所以我們接著檢測 A375 細胞中發生細胞自噬的比例,以辣椒素濃度 100 μM 以上處理細
胞,可發現細胞自噬比例有明顯的提升,而當細胞自噬被抑制時,辣椒素 200 μM細胞凋亡比例增加。進一步我們利用 shRNA 技術抑制 tNOX 蛋白表現,再以辣椒素處理,發現細胞對辣椒素的處理變得較為敏感,在濃度 100 μM 以上細胞凋亡比例就有顯著的提升,這些結果顯示,對於辣椒素處理不敏感的細胞,可藉由抑制細胞自噬或抑制 tNOX 蛋白表現,增加細胞凋亡的發生。

Capsaicin is a major component of the hot chili pepper and believed to be a chemopreventive agent. tNOX, a tumor associated NADH oxidase, is a growth-related protein present in transformed cells. Our previous studies have
showed that high dose of capsaicin exposure not only inhibits tNOX activity and protein expression but also leads to growth inhibition and apoptosis in cancer
cells. However, low concentration of capsaicin treatments (below 10 μM) enhanced tNOX protein expression, cell proliferation, and migration, concurrently with the epithelial-mesenchymal transition. These results demonstrate tNOX protein expression is associated with tumorgenesis. To study the effect of capsaicin on the growth of A375 cells, we exposed A375 cells to capsaicin at different concentrations. Our results showed that exposure of cells to capsaicin at concentration higher than 50 μM inhibits cell growth. Capsaicin induces apoptosis at 400 μM in association with the down-regulation of tNOX protein expression level. At 72 hours, we found that low concentration of capsaicin treatment (10 μM) enhanced A375 cell growth. Due to the fact that autophagy helps cells escape from apoptosis, we speculate whether capsaicin induces autophagy in our system. Our results show that
autophagy is significantly induced in response to 100 μM capsaicin and higher. Furthermore, apoptosis is increased when autophagy is inhibited by 3-MA pretreatment. Moreover, tNOX-shRNA knockdown increases capsaicin-mediated apoptosis in A375 cells. Taken together, our results demonstrate that inhibition of tNOX protein expression or autophagy enhances
apoptosis induction mediated by capsaicin in cancer cells.

目次
中文摘要………………………………………………………………… i
英文摘要……………………………………………………………... ii
目次………………………………………………………………….. iii
第壹章、 前言............................................. 1
一、 惡性腫瘤........................................................ 1
二、 辣椒素 (Capsaicin)................................. 2
三、 Tumor-associated NADH oxidase (tNOX) .............. 4
四、 細胞自噬 (Autophagy) .................................………....... 8
五、 細胞自噬在腫瘤中的重要性…………………….......….. 9
第貳章、 研究動機…………………………………………………… 12
第叁章、 實驗方法…………………………………………………… 13
一、 人類皮膚癌細胞株A375之培養………………………...…… 13
二、 西方墨漬蛋白表現偵測 (Western blotting).……………… 13
三、 細胞DNA轉染技術 (Transfection) …………………………. 14
四、 細胞凋亡測試 (Apoptosis).……………………………………15
五、 細胞自噬測(Autophagy assay)………………………………. 15
六、 過氧化物含量分析 (ROS measurement)..………………….. 16
七、 細胞計數 (Trypan Blue Assay)..……………………….... 16
八、 質體的轉化與純化 (Plasmid transformation and purification)............................................ 16
九、 統計分析 (Statistical analysis)………............... 17
第肆章、 實驗材料…………………………………………………… 18
第伍章、 結果………………………………………………………… 23
一、 不同濃度的辣椒素對A375細胞生長的影響…………………….23
二、 不同濃度辣椒素對A375細胞株細胞毒性……………………… 23
三、 辣椒素對Apoptosis相關蛋白表現量的影響………………… 24
四、 辣椒素對Anti-apoptosis蛋白表現量的影響……………… 24
五、 辣椒素細胞內ROS濃度的影響………………………………… 25
六、 Caspase抑制劑對辣椒素引起細胞凋亡的影響……………… 25
七、 不同濃度辣椒素對tNOX蛋白表現的影響……………………… 25
八、 不同濃度辣椒素處理誘發的細胞自噬反應…………………… 26
九、 抑制細胞自噬對細胞凋亡的影響………………………..... 26
十、 抑制tNOX蛋白表現對於A375細胞株細胞凋亡的影響........ 27
十一、 抑制tNOX蛋白表現對於A375細胞株細胞自噬的影響...... 27
第陸章、 結論……………………………………….……………... 29
第柒章、 討論…………………………………………………….............. 30
第捌章、 參考文獻……………………………….……………..... 33
第玖章、 結果圖表……………………………….……………..... 39



[1] G.P. Guy, D.U. Ekwueme, Years of potential life lost and indirect costs of
melanoma and non-melanoma skin cancer: a systematic review of the literature.
Pharmacoeconomics 29 (2011) 863-874.
[2] H. Sumimoto, M. Miyagishi, H. Miyoshi, S. Yamagata, A. Shimizu, K. Taira, Y.
Kawakami, Inhibition of growth and invasive ability of melanoma by inactivation
of mutated BRAF with lentivirus-mediated RNA interference. Oncogene 23
(2004) 6031-6039.
[3] E. Monaghan-Benson, K. Burridge, Mutant B-RAF regulates a Rac-dependent
cadherin switch in melanoma. Oncogene (2012).
[4] D.J. Slamon, W. Godolphin, L.A. Jones, J.A. Holt, S.G. Wong, D.E. Keith, W.J.
Levin, S.G. Stuart, J. Udove, A. Ullrich, et al., Studies of the HER-2/neu
proto-oncogene in human breast and ovarian cancer. Science 244 (1989) 707-712.
[5] M. Cuello, S.A. Ettenberg, A.S. Clark, M.M. Keane, R.H. Posner, M.M. Nau, P.A.
Dennis, S. Lipkowitz, Down-regulation of the erbB-2 receptor by trastuzumab
(herceptin) enhances tumor necrosis factor-related apoptosis-inducing
ligand-mediated apoptosis in breast and ovarian cancer cell lines that overexpress
erbB-2. Cancer Res 61 (2001) 4892-4900.
[6] D. Veale, T. Ashcroft, C. Marsh, G.J. Gibson, A.L. Harris, Epidermal growth
factor receptors in non-small cell lung cancer. Br J Cancer 55 (1987) 513-516.
[7] J.W. Xu, Q.Q. Li, L.L. Tao, Y.Y. Cheng, J. Yu, Q. Chen, X.P. Liu, Z.D. Xu,
Involvement of EGFR in the promotion of malignant properties in multidrug
resistant breast cancer cells. Int J Oncol 39 (2011) 1501-1509.
[8] K.J. Jeong, K.H. Cho, N. Panupinthu, H. Kim, J. Kang, C.G. Park, G.B. Mills,
H.Y. Lee, EGFR mediates LPA-induced proteolytic enzyme expression and
ovarian cancer invasion: Inhibition by resveratrol. Mol Oncol (2012).
[9] D.J. Morre, P.J. Chueh, D.M. Morre, Capsaicin inhibits preferentially the NADH
oxidase and growth of transformed cells in culture. Proc Natl Acad Sci U S A 92
(1995) 1831-1835.
[10] A. Szallasi, P.M. Blumberg, Vanilloid (Capsaicin) receptors and mechanisms.
Pharmacol Rev 51 (1999) 159-212.
[11] Y. Monsereenusorn, S. Kongsamut, P.D. Pezalla, Capsaicin--a literature survey.
Crit Rev Toxicol 10 (1982) 321-339.
[12] Y.M. Lee, Y.K. Kim, J.H. Chung, Increased expression of TRPV1 channel in
34

intrinsically aged and photoaged human skin in vivo. Exp Dermatol 18 (2009)
431-436.
[13] Y.M. Lee, S.M. Kang, J.H. Chung, The role of TRPV1 channel in aged human
skin. J Dermatol Sci 65 (2012) 81-85.
[14] P. Holzer, Capsaicin: cellular targets, mechanisms of action, and selectivity for
thin sensory neurons. Pharmacol Rev 43 (1991) 143-201.
[15] F. Sicuteri, B.M. Fusco, S. Marabini, V. Campagnolo, C.A. Maggi, P. Geppetti,
M. Fanciullacci, Beneficial effect of capsaicin application to the nasal mucosa in
cluster headache. Clin J Pain 5 (1989) 49-53.
[16] A. Mori, S. Lehmann, J. O''Kelly, T. Kumagai, J.C. Desmond, M. Pervan, W.H.
McBride, M. Kizaki, H.P. Koeffler, Capsaicin, a component of red peppers,
inhibits the growth of androgen-independent, p53 mutant prostate cancer cells.
Cancer Res 66 (2006) 3222-3229.
[17] C.S. Kim, W.H. Park, J.Y. Park, J.H. Kang, M.O. Kim, T. Kawada, H. Yoo, I.S.
Han, R. Yu, Capsaicin, a spicy component of hot pepper, induces apoptosis by
activation of the peroxisome proliferator-activated receptor gamma in HT-29
human colon cancer cells. J Med Food 7 (2004) 267-273.
[18] Y.C. Lo, Y.C. Yang, I.C. Wu, F.C. Kuo, C.M. Liu, H.W. Wang, C.H. Kuo, J.Y.
Wu, D.C. Wu, Capsaicin-induced cell death in a human gastric adenocarcinoma
cell line. World J Gastroenterol 11 (2005) 6254-6257.
[19] M.Y. Jung, H.J. Kang, A. Moon, Capsaicin-induced apoptosis in SK-Hep-1
hepatocarcinoma cells involves Bcl-2 downregulation and caspase-3 activation.
Cancer Lett 165 (2001) 139-145.
[20] K. Ito, T. Nakazato, K. Yamato, Y. Miyakawa, T. Yamada, N. Hozumi, K.
Segawa, Y. Ikeda, M. Kizaki, Induction of apoptosis in leukemic cells by
homovanillic acid derivative, capsaicin, through oxidative stress: implication of
phosphorylation of p53 at Ser-15 residue by reactive oxygen species. Cancer
Res 64 (2004) 1071-1078.
[21] M. Bhutani, A.K. Pathak, A.S. Nair, A.B. Kunnumakkara, S. Guha, G. Sethi,
B.B. Aggarwal, Capsaicin is a novel blocker of constitutive and
interleukin-6-inducible STAT3 activation. Clin Cancer Res 13 (2007)
3024-3032.
[22] S.H. Lee, R.L. Richardson, R.H. Dashwood, S.J. Baek, Capsaicin represses
transcriptional activity of beta-catenin in human colorectal cancer cells. J Nutr
Biochem 23 (2012) 646-655.
[23] S. Malagarie-Cazenave, N. Olea-Herrero, D. Vara, C. Morell, I. Diaz-Laviada,
The vanilloid capsaicin induces IL-6 secretion in prostate PC-3 cancer cells.
35

Cytokine 54 (2011) 330-337.
[24] C.C. Wu, J.P. Lin, J.S. Yang, S.T. Chou, S.C. Chen, Y.T. Lin, H.L. Lin, J.G.
Chung, Capsaicin induced cell cycle arrest and apoptosis in human esophagus
epidermoid carcinoma CE 81T/VGH cells through the elevation of intracellular
reactive oxygen species and Ca2+ productions and caspase-3 activation. Mutat
Res 601 (2006) 71-82.
[25] S.Y. Lee, Y. Hong, U. Oh, Decreased pain sensitivity of capsaicin-treated rats
results from decreased VR1 expression. Arch Pharm Res 27 (2004) 1154-1160.
[26] A.O. Brightman, J. Wang, R.K. Miu, I.L. Sun, R. Barr, F.L. Crane, D.J. Morre, A
growth factor- and hormone-stimulated NADH oxidase from rat liver plasma
membrane. Biochim Biophys Acta 1105 (1992) 109-117.
[27] M. Bruno, A.O. Brightman, J. Lawrence, D. Werderitsh, D.M. Morre, D.J.
Morre, Stimulation of NADH oxidase activity from rat liver plasma membranes
by growth factors and hormones is decreased or absent with hepatoma plasma
membranes. Biochem J 284 ( Pt 3) (1992) 625-628.
[28] H.M. Wang, S.M. Chuang, Y.C. Su, Y.H. Li, P.J. Chueh, Down-regulation of
tumor-associated NADH oxidase, tNOX (ENOX2), enhances capsaicin-induced
inhibition of gastric cancer cell growth. Cell Biochem Biophys 61 (2011)
355-366.
[29] P.J. Chueh, C. Kim, N. Cho, D.M. Morre, D.J. Morre, Molecular cloning and
characterization of a tumor-associated, growth-related, and time-keeping
hydroquinone (NADH) oxidase (tNOX) of the HeLa cell surface. Biochemistry
41 (2002) 3732-3741.
[30] I.L. Sun, E.E. Sun, F.L. Crane, D.J. Morre, A. Lindgren, H. Low, Requirement
for coenzyme Q in plasma membrane electron transport. Proc Natl Acad Sci U S
A 89 (1992) 11126-11130.
[31] F. Wilkinson, C. Kim, N. Cho, P.J. Chueh, S. Leslie, S. Moya-Camarena, L.Y.
Wu, D.M. Morre, D.J. Morre, Isolation and identification of a protein with
capsaicin-inhibited NADH oxidase activity from culture media conditioned by
growth of HeLa cells. Arch Biochem Biophys 336 (1996) 275-282.
[32] D.J. Morre, S. Caldwell, A. Mayorga, L.Y. Wu, D.M. Morre, NADH oxidase
activity from sera altered by capsaicin is widely distributed among cancer
patients. Arch Biochem Biophys 342 (1997) 224-230.
[33] P.J. Chueh, L.Y. Wu, D.M. Morre, D.J. Morre, tNOX is both necessary and
sufficient as a cellular target for the anticancer actions of capsaicin and the green
tea catechin (-)-epigallocatechin-3-gallate. Biofactors 20 (2004) 235-249.
[34] L.C. Mao, H.M. Wang, Y.Y. Lin, T.K. Chang, Y.H. Hsin, P.J. Chueh,
36

Stress-induced down-regulation of tumor-associated NADH oxidase during
apoptosis in transformed cells. FEBS Lett 582 (2008) 3445-3450.
[35] K. Yagiz, D.J. Morre, D.M. Morre, Transgenic mouse line overexpressing the
cancer-specific tNOX protein has an enhanced growth and acquired
drug-response phenotype. J Nutr Biochem 17 (2006) 750-759.
[36] M.K. Hwang, A.M. Bode, S. Byun, N.R. Song, H.J. Lee, K.W. Lee, Z. Dong,
Cocarcinogenic effect of capsaicin involves activation of EGFR signaling but
not TRPV1. Cancer Res 70 (2010) 6859-6869.
[37] N. Erin, W. Zhao, J. Bylander, G. Chase, G. Clawson, Capsaicin-induced
inactivation of sensory neurons promotes a more aggressive gene expression
phenotype in breast cancer cells. Breast Cancer Res Treat 99 (2006) 351-364.
[38] N.C. Liu, P.F. Hsieh, M.K. Hsieh, Z.M. Zeng, H.L. Cheng, J.W. Liao, P.J. Chueh,
Capsaicin-mediated tNOX (ENOX2) up-regulation enhances cell proliferation
and migration in vitro and in vivo. J Agric Food Chem 60 (2012) 2758-2765.
[39] Z.M. Zeng, S.M. Chuang, T.C. Chang, C.W. Hong, J.C. Chou, J.J. Yang, P.J.
Chueh, Phosphorylation of serine-504 of tNOX (ENOX2) modulates cell
proliferation and migration in cancer cells. Exp Cell Res 318 (2012) 1759-1766.
[40] S.H. Oh, Y.S. Kim, S.C. Lim, Y.F. Hou, I.Y. Chang, H.J. You, Dihydrocapsaicin
(DHC), a saturated structural analog of capsaicin, induces autophagy in human
cancer cells in a catalase-regulated manner. Autophagy 4 (2008) 1009-1019.
[41] T. Yorimitsu, D.J. Klionsky, Autophagy: molecular machinery for self-eating.
Cell Death Differ 12 Suppl 2 (2005) 1542-1552.
[42] W. Bursch, The autophagosomal-lysosomal compartment in programmed cell
death. Cell Death Differ 8 (2001) 569-581.
[43] G. Kroemer, M. Jaattela, Lysosomes and autophagy in cell death control. Nat
Rev Cancer 5 (2005) 886-897.
[44] E.H. Baehrecke, Autophagy: dual roles in life and death? Nat Rev Mol Cell Biol
6 (2005) 505-510.
[45] D.J. Klionsky, S.D. Emr, Autophagy as a regulated pathway of cellular
degradation. Science 290 (2000) 1717-1721.
[46] X.H. Ma, S. Piao, D. Wang, Q.W. McAfee, K.L. Nathanson, J.J. Lum, L.Z. Li,
R.K. Amaravadi, Measurements of tumor cell autophagy predict invasiveness,
resistance to chemotherapy, and survival in melanoma. Clin Cancer Res 17
(2011) 3478-3489.
[47] B.G. Wouters, T. van den Beucken, M.G. Magagnin, P. Lambin, C. Koumenis,
Targeting hypoxia tolerance in cancer. Drug Resist Updat 7 (2004) 25-40.
[48] M.Z. Noman, B. Janji, B. Kaminska, K. Van Moer, S. Pierson, P. Przanowski, S.
37

Buart, G. Berchem, P. Romero, F. Mami-Chouaib, S. Chouaib, Blocking
hypoxia-induced autophagy in tumors restores cytotoxic T-cell activity and
promotes regression. Cancer Res 71 (2011) 5976-5986.
[49] R. Mathew, C.M. Karp, B. Beaudoin, N. Vuong, G. Chen, H.Y. Chen, K. Bray, A.
Reddy, G. Bhanot, C. Gelinas, R.S. Dipaola, V. Karantza-Wadsworth, E. White,
Autophagy suppresses tumorigenesis through elimination of p62. Cell 137 (2009)
1062-1075.
[50] G. Kroemer, B. Levine, Autophagic cell death: the story of a misnomer. Nat Rev
Mol Cell Biol 9 (2008) 1004-1010.
[51] N. Mizushima, B. Levine, A.M. Cuervo, D.J. Klionsky, Autophagy fights
disease through cellular self-digestion. Nature 451 (2008) 1069-1075.
[52] S. Yoon, S.U. Woo, J.H. Kang, K. Kim, M.H. Kwon, S. Park, H.J. Shin, H.S.
Gwak, Y.J. Chwae, STAT3 transcriptional factor activated by reactive oxygen
species induces IL6 in starvation-induced autophagy of cancer cells. Autophagy
6 (2010) 1125-1138.
[53] M.A. de la Cruz-Morcillo, M.L. Valero, J.L. Callejas-Valera, L. Arias-Gonzalez,
P. Melgar-Rojas, E.M. Galan-Moya, E. Garcia-Gil, J. Garcia-Cano, R.
Sanchez-Prieto, P38MAPK is a major determinant of the balance between
apoptosis and autophagy triggered by 5-fluorouracil: implication in resistance.
Oncogene 31 (2012) 1073-1085.
[54] N.H. Thoennissen, J. O''Kelly, D. Lu, G.B. Iwanski, D.T. La, S. Abbassi, A.
Leiter, B. Karlan, R. Mehta, H.P. Koeffler, Capsaicin causes cell-cycle arrest and
apoptosis in ER-positive and -negative breast cancer cells by modulating the
EGFR/HER-2 pathway. Oncogene 29 (2010) 285-296.
[55] C.H. Choi, Y.K. Jung, S.H. Oh, Autophagy induction by capsaicin in malignant
human breast cells is modulated by p38 and extracellular signal-regulated
mitogen-activated protein kinases and retards cell death by suppressing
endoplasmic reticulum stress-mediated apoptosis. Mol Pharmacol 78 (2010)
114-125.
[56] J.H. Yoon, S.G. Ahn, B.H. Lee, S.H. Jung, S.H. Oh, Role of autophagy in
chemoresistance: regulation of the ATM-mediated DNA-damage signaling
pathway through activation of DNA-PKcs and PARP-1. Biochem Pharmacol 83
(2012) 747-757.
[57] D. Hanahan, R.A. Weinberg, Hallmarks of cancer: the next generation. Cell 144
(2011) 646-674.
[58] T. Kurita, Y.Z. Wang, A.A. Donjacour, C. Zhao, J.P. Lydon, B.W. O''Malley, J.T.
Isaacs, R. Dahiya, G.R. Cunha, Paracrine regulation of apoptosis by steroid
38

hormones in the male and female reproductive system. Cell Death Differ 8
(2001) 192-200.
[59] M.J. Abedin, D. Wang, M.A. McDonnell, U. Lehmann, A. Kelekar, Autophagy
delays apoptotic death in breast cancer cells following DNA damage. Cell Death
Differ 14 (2007) 500-510.
[60] S. Kongara, V. Karantza, The interplay between autophagy and ROS in
tumorigenesis. Front Oncol 2 (2012) 171.
[61] A. Puissant, N. Fenouille, P. Auberger, When autophagy meets cancer through
p62/SQSTM1. Am J Cancer Res 2 (2012) 397-413.
[62] J. Moscat, M.T. Diaz-Meco, p62: a versatile multitasker takes on cancer. Trends
Biochem Sci 37 (2012) 230-236.
[63] H. Kanamori, G. Takemura, K. Goto, R. Maruyama, A. Tsujimoto, A. Ogino, T.
Takeyama, T. Kawaguchi, T. Watanabe, T. Fujiwara, H. Fujiwara, M. Seishima,
S. Minatoguchi, The role of autophagy emerging in postinfarction cardiac
remodelling. Cardiovasc Res 91 (2011) 330-339.
[64] J.F. Rivera, T. Gurlo, M. Daval, C.J. Huang, A.V. Matveyenko, P.C. Butler, S.
Costes, Human-IAPP disrupts the autophagy/lysosomal pathway in pancreatic
beta-cells: protective role of p62-positive cytoplasmic inclusions. Cell Death
Differ 18 (2011) 415-426.

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