研究一：藉由蛋白質體學分析高糖環境下人類肝臟細胞內蛋白質體變化及其參與糖尿病誘導肝臟疾病之相關機制

在高血糖下會造成肝臟細胞功能失調而引起許多代謝相關的疾病，其中包括了糖尿病型肝病。過去許多研究指出，高血糖會經由glycoxidation而導致肝臟細胞的損傷，然而，高血糖造成的這些效應的機制至今仍尚未被詳盡地研究出。在本研究中，我們將Chang liver cell分別培養在5.5 mM、25 mM 及 100 mM三種不同濃度葡萄糖培養液中，並利用甘露醇調整其滲透壓，進而利用蛋白質體學的方式研究這三種不同糖濃度培養下的細胞中其蛋白質表現差異及氧化還原的調節。在本研究結果中，我們鑑定到了在不同糖濃度培養下的細胞中，141個蛋白質具有表現量差異、29個蛋白質具有巰基變化。這些具有表現量差異的蛋白質參與了轉錄的調控、訊號傳遞、氧化還原的調控、細胞骨架的調控等功能，而具有巰基變化的蛋白質則是參與了蛋白質摺疊與基因調控等功能。我們進一步利用了臨床上的糖尿病病患與健康捐贈者的血清檢體確認galectin-3、GRP-78、GSTP1等蛋白質在血清中的表現量差異，以驗證蛋白質體學實驗中所得到的結果。總歸來說，本研究中我們利用完善的蛋白質體學平台分析高糖濃度下培養的肝臟細胞中，其蛋白質表現量差異以及巰基的變化，鑑定出了與糖尿病型肝病之病程發展相關的蛋白質，並且利用臨床檢體進行進一步的確認。我們認為，這些鑑定出的蛋白質未來在糖尿病型肝病的預測與診斷當中，將會成為非常有潛力的生物分子標靶。


研究二：藉由蛋白質體學分析具有pemetrexed抗藥性之人類肺腺癌細胞內蛋白質體變化及參與之相關機制

目前世界衛生組織統計資料中顯示，肺癌是全球十大死因之一，而在國內更是位居癌症死亡率之首，由此可見肺癌的治療與預防在現今的社會當中是相當迫切且不容小覷的。雖然目前肺癌用藥的研究與發展已相當蓬勃，但在臨床上的使用仍有很大的限制，其中一個主要的原因就是伴隨著療程時間越久而日趨嚴重的抗藥性問題。肺腺癌是所有肺癌種類中的最大宗，所佔的比例高達百分之四十，而pemetrexed是臨床上治療肺腺癌的第一線用藥之一。
在本研究中，我們使用A549與A549/PEM兩株分別對pemetrexed敏感以及具有抗性的細胞株，利用蛋白質體學的方式研究這兩種不同特性的細胞其蛋白質表現差異以及可能參與的抗藥性機制。在本研究結果中，我們鑑定到在A549處理pemetrexed之後，有81個蛋白質具有顯著的表現量差異，但在處理pemetrexed的A549/PEM中卻沒有顯著的變化；反之，在皆沒有處理pemetrexed的情況下，A549與A549/PEM間有72個蛋白質具有顯著的表現量差異，而這些具有表現量差異的蛋白可協助我們進一步探究造成pemetrexed抗藥性的相關機制。
此外，我利用siRNA干擾技術將Calreticulin、Flavin reductase以及Progesterone receptor component 1三個蛋白的表現抑制後，發現可以有效地抑制掉A549/PEM細胞所具有的抗藥性，降低細胞的存活率以及增加細胞凋亡。我們認為，這些鑑定出來在抗藥性細胞中具有差異表現的蛋白質，未來在pemetrexed抗藥性疾病的診斷當中，將會成為非常有潛力的生物分子標靶。

Project I: High glucose-induced proteome alterations in hepatocytes and its possible relevance to diabetic liver disease

Hyperglycemia can cause a variety of abnormal disorders in liver cells, one of these abnormalities is diabetic liver disease. Previous study has shown that high glucose concentration in blood can damage liver cells via glycoxidation. However, the details of molecular mechanisms underlying the effects of high glucose concentration in blood in the development of diabetic liver disease have not to be elucidated. In this study, we incubated a liver cell line (Chang liver cell) in mannitol-balanced 5.5 mM, 25 mM and 100 mM glucose media and assessed protein expressional levels and redox-regulations. We identified 141 proteins that showed significant alterations in protein expression and 29 proteins that showed significant alterations in thiol reactivity in response to high glucose concentration. According to the proteomic results, several proteins involved in transcription-control, signal transduction, redox regulation, and cytoskeleton regulation have showed significant alterations in expression. However, proteins involved in protein folding and gene regulation have displayed significant alterations in redox regulations, related to the thiol reactivity. Further analyses by utilizing clinical plasma specimens confirmed that the proteins showed type 2 diabetic liver disease-dependent changes, such as galectin-3, GRP-78, GSTP1, etc. In conclusion, in this study we used a comprehensive hepatocyte-based proteomic approach to identify the high glucose concentration-induced alterations in protein expressional level and to identify redox-associated diabetic liver disease markers. Several identified proteins were validated with clinical samples and might serve as potential targets for the prognosis and diagnosis of diabetic liver disease.


Project II: Proteomic analysis of proteins responsible for the development of pemetrexed resistance in human lung adenocarcinoma

Lung cancer occupies the top 7 leading causes of death in the world and is the top one deadliest cancer in domestic. However, cancer drug resistance is one of the major reasons which cause the failure of chemotherapy in lung cancer. To comprehend the more detailed mechanisms of drug resistance for lung cancer, we used pemetrexed-sensitive-A549 cells and pemetrexed-resistant A549/PEM cells to examine the pemetrexed-resistance-dependent cellular responses and to identify the potential therapeutic targets for drug resistance. We combined two-dimensional differential gel electrophoresis (2D-DIGE) and matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) to investigate the global protein expression alterations induced by pemetrexed treatment and pemetrexed resistance. A proteomic study indicated that pemetrexed-exposure changed the expressions of 81 proteins in A549 cells, whereas no significant response took place in treated A549/PEM cells, indicating these proteins are associated with drug specific resistance. Moreover, 72 proteins demonstrated differentially expressional levels between A549 cells and A549/PEM cells regarding as baseline resistance.
Further studies have used siRNA silencing to against calreticulin, flavin reductase and membrane-associated progesterone receptor component 1 (PGRMC1) proteins, to examine and evaluate their potency in the formation of pemetrexed resistance. The proteomic approach allowed us to identify numerous proteins which are involved in a variety of drug-resistance-forming mechanisms. In this study, we provide useful therapeutic candidates and diagnostic markers for the treatment of pemetrexed-resistant lung cancer.

研究一：中文摘要 I
研究二：中文摘要 II
Project I: ABSTRACT III
Project II: ABSTRACT IV
誌謝 V
TABLE OF CONTENTS VII
LIST OF FIGURES AND TABLES X
ABBREVIATIONS XV

Project I: 1
Chapter 1 INTRODUCTION 2
1.1 Diabetes mellitus 2
1.2 High glucose-induced cell response 4
1.3 Diabetic liver disease 5
1.4 Overview on proteomics 6
1.5 Aim of this study 17
Chapter 2 MATERIALS AND METHODS 18
2.1 Chemicals and reagents 18
2.2 Cell lines and cell cultures 19
2.3 Functional assays 20
2.4 Proteomic strategies 22
2.5 Validation of identified proteins 31
Chapter 3 RESULTS 35
3.1 Effect of glucose concentration on cell viability and cell apoptosis in Chang liver cells. 35
3.2 Comparison of protein expression levels between different glucose concentrations cultured Chang liver cells by lysine labeling 2D-DIGE analysis 38
3.3 Identification of differentially expressed proteins in 2D-DIGE by using MALDI-TOF-MS analysis 39
3.4 Validation of differentially expressed proteins identified through expression proteomic study in Chang liver cells incubated in 5.5 mM, 25mM and 100 mM glucose 47
3.5 Validation of differentially expressed proteins identified through expression proteomic study in plasma for Type 2 DM patients and healthy donors 52
3.6 Redox proteomic analysis of high glucose-induced cysteine modifications in Chang liver cell proteins 58
3.7 Identification of thiol reactive proteins using MALDI-TOF-MS analysis 61
3.8 Effect of hyperglycemia-induced phosphorylation of protein kinase C 64
Chapter 4 DISCUSSIONS 66
Chapter 5 CONCLUSIONS 75
Chapter 6 REFERENCE 77

Project II: 81
Chapter 1 INTRODUCTION 82
1.1 Lung cancer 82
1.2 Cancer drug resistance 87
1.3 Pemetrexed 91
1.4 Small interfering RNAs for gene silencing 95
1.5 Aim of this study 97
Chapter 2 MATERIALS AND METHODS 98
2.1 Chemicals and reagents 98
2.2 Cell lines and cell cultures 99
2.3 MTT cell viability assay 100
2.4 Proteomic strategies 100
2.5 Western blotting analysis 100
2.6 Flow cytometry analysis for apoptosis detection 100
2.7 siRNA design, construction and transfection 102
Chapter 3 RESULTS 104
3.1 Pemetrexed-induced loss of cell viability on A549 and A549/PEM cells 104
3.2 Comparison of protein expression levels between baseline and pemetrexed specific resistance in human lung adenocarcinoma by 2D-DIGE analysis 105
3.3 Identification of differentially expressed proteins in 2D-DIGE by using MALDI-TOF-MS analysis 110
3.3 Validation of characterized baseline resistance and pemetrexed specific response associated proteins via western blotting analysis 118
3.4 Evaluation of the roles of calreticulin and flavin reductase on pemetrexed resistance in lung adenocarcinoma using siRNA knockdown 121
3.5 Evaluation of the roles of PGRMC1 on pemetrexed resistance in lung adenocarcinoma 129
Chapter 4 DISCUSSIONS 134
Chapter 5 CONCLUSIONS 144
Chapter 6 REFERENCE 145

APPENDIX i
Supplementary table 1 ii
Supplementary table 2 xvi
Supplementary table 3 xviii
High glucose-induced proteome alterations in hepatocytes and its possible relevance to diabetic liver disease.

Project I:
[1] WHO. The Top 10 Causes of Death. World Health Organisation; 2013.
[2] Gillery P. [Oxidative stress and protein glycation in diabetes mellitus]. Ann Biol Clin (Paris). 2006;64:309-14.
[3] Goldin A, Beckman JA, Schmidt AM, Creager MA. Advanced glycation end products: sparking the development of diabetic vascular injury. Circulation. 2006;114:597-605.
[4] Brownlee M. Advanced protein glycosylation in diabetes and aging. Annual review of medicine. 1995;46:223-34.
[5] Striker LJ, Striker GE. Administration of AGEs in vivo induces extracellular matrix gene expression. Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association. 1996;11 Suppl 5:62-5.
[6] Hofmann MA, Drury S, Fu C, Qu W, Taguchi A, Lu Y, et al. RAGE mediates a novel proinflammatory axis: a central cell surface receptor for S100/calgranulin polypeptides. Cell. 1999;97:889-901.
[7] Yang H, Jin X, Kei Lam CW, Yan SK. Oxidative stress and diabetes mellitus. Clinical chemistry and laboratory medicine : CCLM / FESCC. 2011;49:1773-82.
[8] Nakajima K, Yamauchi K, Shigematsu S, Ikeo S, Komatsu M, Aizawa T, et al. Selective attenuation of metabolic branch of insulin receptor down-signaling by high glucose in a hepatoma cell line, HepG2 cells. The Journal of biological chemistry. 2000;275:20880-6.
[9] Sauvaget D, Chauffeton V, Dugue-Pujol S, Kalopissis AD, Guillet-Deniau I, Foufelle F, et al. In vitro transcriptional induction of the human apolipoprotein A-II gene by glucose. Diabetes. 2004;53:672-8.
[10] Zang M, Zuccollo A, Hou X, Nagata D, Walsh K, Herscovitz H, et al. AMP-activated protein kinase is required for the lipid-lowering effect of metformin in insulin-resistant human HepG2 cells. The Journal of biological chemistry. 2004;279:47898-905.
[11] Tolman KG, Fonseca V, Dalpiaz A, Tan MH. Spectrum of liver disease in type 2 diabetes and management of patients with diabetes and liver disease. Diabetes care. 2007;30:734-43.
[12] Trombetta M, Spiazzi G, Zoppini G, Muggeo M. Review article: type 2 diabetes and chronic liver disease in the Verona diabetes study. Alimentary pharmacology & therapeutics. 2005;22 Suppl 2:24-7.
[13] Belcher G, Schernthaner G. Changes in liver tests during 1-year treatment of patients with Type 2 diabetes with pioglitazone, metformin or gliclazide. Diabetic medicine : a journal of the British Diabetic Association. 2005;22:973-9.
[14] Lebovitz HE, Kreider M, Freed MI. Evaluation of liver function in type 2 diabetic patients during clinical trials: evidence that rosiglitazone does not cause hepatic dysfunction. Diabetes care. 2002;25:815-21.
[15] Baig NA, Herrine SK, Rubin R. Liver disease and diabetes mellitus. Clinics in laboratory medicine. 2001;21:193-207.
[16] Knobler H, Stagnaro-Green A, Wallenstein S, Schwartz M, Roman SH. Higher incidence of diabetes in liver transplant recipients with hepatitis C. Journal of clinical gastroenterology. 1998;26:30-3.
[17] Kawaguchi T, Yoshida T, Harada M, Hisamoto T, Nagao Y, Ide T, et al. Hepatitis C virus down-regulates insulin receptor substrates 1 and 2 through up-regulation of suppressor of cytokine signaling 3. The American journal of pathology. 2004;165:1499-508.
[18] Moscatiello S, Manini R, Marchesini G. Diabetes and liver disease: an ominous association. Nutrition, metabolism, and cardiovascular diseases : NMCD. 2007;17:63-70.
[19] Wilkins MR, Pasquali C, Appel RD, Ou K, Golaz O, Sanchez JC, et al. From proteins to proteomes: large scale protein identification by two-dimensional electrophoresis and amino acid analysis. Bio/technology. 1996;14:61-5.
[20] James P. Protein identification in the post-genome era: the rapid rise of proteomics. Quarterly reviews of biophysics. 1997;30:279-331.
[21] O'Farrell PH. High resolution two-dimensional electrophoresis of proteins. The Journal of biological chemistry. 1975;250:4007-21.
[22] Stochaj WR, Berkelman T, Laird N. Preparative 2D Gel Electrophoresis with Immobilized pH Gradients: Rehydration of IPG Strips for Isoelectric Focusing of Proteins. CSH protocols. 2006;2006.
[23] Kerenyi L, Gallyas F. [Errors in quantitative estimations on agar electrophoresis using silver stain]. Clinica chimica acta; international journal of clinical chemistry. 1973;47:425-36.
[24] Unlu M, Morgan ME, Minden JS. Difference gel electrophoresis: a single gel method for detecting changes in protein extracts. Electrophoresis. 1997;18:2071-7.
[25] Chan HL, Gharbi S, Gaffney PR, Cramer R, Waterfield MD, Timms JF. Proteomic analysis of redox- and ErbB2-dependent changes in mammary luminal epithelial cells using cysteine- and lysine-labelling two-dimensional difference gel electrophoresis. Proteomics. 2005;5:2908-26.
[26] Alban A, David SO, Bjorkesten L, Andersson C, Sloge E, Lewis S, et al. A novel experimental design for comparative two-dimensional gel analysis: two-dimensional difference gel electrophoresis incorporating a pooled internal standard. Proteomics. 2003;3:36-44.
[27] Marouga R, David S, Hawkins E. The development of the DIGE system: 2D fluorescence difference gel analysis technology. Analytical and bioanalytical chemistry. 2005;382:669-78.
[28] Camp Hod. MALDI-TOF.
[29] Chang RS. Continuous subcultivation of epithelial-like cells from normal human tissues. Proceedings of the Society for Experimental Biology and Medicine Society for Experimental Biology and Medicine. 1954;87:440-3.
[30] Nelson-Rees WA, Flandermeyer RR. HeLa cultures defined. Science. 1976;191:96-8.
[31] Candiloros H, Muller S, Zeghari N, Donner M, Drouin P, Ziegler O. Decreased erythrocyte membrane fluidity in poorly controlled IDDM. Influence of ketone bodies. Diabetes care. 1995;18:549-51.
[32] Saydah SH, Miret M, Sung J, Varas C, Gause D, Brancati FL. Postchallenge hyperglycemia and mortality in a national sample of U.S. adults. Diabetes care. 2001;24:1397-402.
[33] Cai L, Li W, Wang G, Guo L, Jiang Y, Kang YJ. Hyperglycemia-induced apoptosis in mouse myocardium: mitochondrial cytochrome C-mediated caspase-3 activation pathway. Diabetes. 2002;51:1938-48.
[34] Haller H, Baur E, Quass P, Behrend M, Lindschau C, Distler A, et al. High glucose concentrations and protein kinase C isoforms in vascular smooth muscle cells. Kidney international. 1995;47:1057-67.
[35] Hsieh PS, Hsieh YJ. Impact of liver diseases on the development of type 2 diabetes mellitus. World journal of gastroenterology : WJG. 2011;17:5240-5.
[36] Ghezzi P, Bonetto V, Fratelli M. Thiol-disulfide balance: from the concept of oxidative stress to that of redox regulation. Antioxidants & redox signaling. 2005;7:964-72.
[37] Giacco F, Brownlee M. Oxidative stress and diabetic complications. Circulation research. 2010;107:1058-70.
[38] Chen YH, Chen JY, Chen YW, Lin ST, Chan HL. High glucose-induced proteome alterations in retinal pigmented epithelium cells and its possible relevance to diabetic retinopathy. Molecular bioSystems. 2012;8:3107-24.
[39] Negre-Salvayre A, Salvayre R, Auge N, Pamplona R, Portero-Otin M. Hyperglycemia and glycation in diabetic complications. Antioxidants & redox signaling. 2009;11:3071-109.
[40] Vikramadithyan RK, Hu Y, Noh HL, Liang CP, Hallam K, Tall AR, et al. Human aldose reductase expression accelerates diabetic atherosclerosis in transgenic mice. The Journal of clinical investigation. 2005;115:2434-43.
[41] Srivastava SK, Ramana KV, Bhatnagar A. Role of aldose reductase and oxidative damage in diabetes and the consequent potential for therapeutic options. Endocrine reviews. 2005;26:380-92.
[42] Hayes JD, McLellan LI. Glutathione and glutathione-dependent enzymes represent a co-ordinately regulated defence against oxidative stress. Free radical research. 1999;31:273-300.
[43] Amer MA, Ghattas MH, Abo-Elmatty DM, Abou-El-Ela SH. Influence of glutathione S-transferase polymorphisms on type-2 diabetes mellitus risk. Genetics and molecular research : GMR. 2011;10:3722-30.
[44] Allenby G, Bocquel MT, Saunders M, Kazmer S, Speck J, Rosenberger M, et al. Retinoic acid receptors and retinoid X receptors: interactions with endogenous retinoic acids. Proceedings of the National Academy of Sciences of the United States of America. 1993;90:30-4.
[45] Singh AB, Guleria RS, Nizamutdinova IT, Baker KM, Pan J. High glucose-induced repression of RAR/RXR in cardiomyocytes is mediated through oxidative stress/JNK signaling. Journal of cellular physiology. 2012;227:2632-44.
[46] Guleria RS, Choudhary R, Tanaka T, Baker KM, Pan J. Retinoic acid receptor-mediated signaling protects cardiomyocytes from hyperglycemia induced apoptosis: role of the renin-angiotensin system. Journal of cellular physiology. 2011;226:1292-307.
[47] Zhong Y, Li J, Chen Y, Wang JJ, Ratan R, Zhang SX. Activation of endoplasmic reticulum stress by hyperglycemia is essential for Muller cell-derived inflammatory cytokine production in diabetes. Diabetes. 2012;61:492-504.
[48] Bhandary B, Marahatta A, Kim HR, Chae HJ. An involvement of oxidative stress in endoplasmic reticulum stress and its associated diseases. International journal of molecular sciences. 2012;14:434-56.
[49] Mori K. [Cellular response to endoplasmic reticulum stress mediated by unfolded protein response pathway]. Tanpakushitsu kakusan koso Protein, nucleic acid, enzyme. 1999;44:2442-8.
[50] Sadighi Akha AA, Harper JM, Salmon AB, Schroeder BA, Tyra HM, Rutkowski DT, et al. Heightened induction of proapoptotic signals in response to endoplasmic reticulum stress in primary fibroblasts from a mouse model of longevity. The Journal of biological chemistry. 2011;286:30344-51.
[51] Yamagishi N, Ueda T, Mori A, Saito Y, Hatayama T. Decreased expression of endoplasmic reticulum chaperone GRP78 in liver of diabetic mice. Biochemical and biophysical research communications. 2012;417:364-70.
[52] Taniguchi CM, Emanuelli B, Kahn CR. Critical nodes in signalling pathways: insights into insulin action. Nature reviews Molecular cell biology. 2006;7:85-96.
[53] Dumic J, Dabelic S, Flogel M. Galectin-3: an open-ended story. Biochimica et biophysica acta. 2006;1760:616-35.
[54] Saksida T, Nikolic I, Vujicic M, Nilsson UJ, Leffler H, Lukic ML, et al. Galectin-3 deficiency protects pancreatic islet cells from cytokine-triggered apoptosis in vitro. Journal of cellular physiology. 2013;228:1568-76.


Project II:
[1] WHO. The Top 10 Causes of Death. World Health Organisation; 2013.
[2] 中華民國衛生福利部. 民國100年癌症登記報告. 2014.
[3] Merck. Lung Carcinoma: Tumors of the Lungs. 2007.
[4] Kenfield SA, Wei EK, Stampfer MJ, Rosner BA, Colditz GA. Comparison of aspects of smoking among the four histological types of lung cancer. Tob Control. 2008;17:198-204.
[5] Subramanian J, Govindan R. Lung cancer in never smokers: a review. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 2007;25:561-70.
[6] Vazquez M, Carter D, Brambilla E, Gazdar A, Noguchi M, Travis WD, et al. Solitary and multiple resected adenocarcinomas after CT screening for lung cancer: histopathologic features and their prognostic implications. Lung cancer. 2009;64:148-54.
[7] Lung cancer staging. Free to breathe; 2014.
[8] Meschini S, Calcabrini A, Monti E, Del Bufalo D, Stringaro A, Dolfini E, et al. Intracellular P-glycoprotein expression is associated with the intrinsic multidrug resistance phenotype in human colon adenocarcinoma cells. International journal of cancer Journal international du cancer. 2000;87:615-28.
[9] Holohan C, Van Schaeybroeck S, Longley DB, Johnston PG. Cancer drug resistance: an evolving paradigm. Nature reviews Cancer. 2013;13:714-26.
[10] Longley DB, Harkin DP, Johnston PG. 5-fluorouracil: mechanisms of action and clinical strategies. Nature reviews Cancer. 2003;3:330-8.
[11] Lin ST, Chou HC, Chang SJ, Chen YW, Lyu PC, Wang WC, et al. Proteomic analysis of proteins responsible for the development of doxorubicin resistance in human uterine cancer cells. Journal of proteomics. 2012;75:5822-47.
[12] Wang Y, Zhao R, Goldman ID. Decreased expression of the reduced folate carrier and folypolyglutamate synthetase is the basis for acquired resistance to the pemetrexed antifolate (LY231514) in an L1210 murine leukemia cell line. Biochemical pharmacology. 2003;65:1163-70.
[13] Diasio RB, Johnson MR. Dihydropyrimidine dehydrogenase: its role in 5-fluorouracil clinical toxicity and tumor resistance. Clinical cancer research : an official journal of the American Association for Cancer Research. 1999;5:2672-3.
[14] Ladner RD. The role of dUTPase and uracil-DNA repair in cancer chemotherapy. Curr Protein Pept Sci. 2001;2:361-70.
[15] Sturm I, Bosanquet AG, Hermann S, Guner D, Dorken B, Daniel PT. Mutation of p53 and consecutive selective drug resistance in B-CLL occurs as a consequence of prior DNA-damaging chemotherapy. Cell death and differentiation. 2003;10:477-84.
[16] Lavarino C, Delia D, Di Palma S, Zunino F, Pilotti S. p53 in drug resistance in ovarian cancer. Lancet. 1997;349:1556.
[17] Breen L, Heenan M, Amberger-Murphy V, Clynes M. Investigation of the role of p53 in chemotherapy resistance of lung cancer cell lines. Anticancer research. 2007;27:1361-4.
[18] Longley DB, Boyer J, Allen WL, Latif T, Ferguson PR, Maxwell PJ, et al. The role of thymidylate synthase induction in modulating p53-regulated gene expression in response to 5-fluorouracil and antifolates. Cancer research. 2002;62:2644-9.
[19] Elsaleh H, Powell B, McCaul K, Grieu F, Grant R, Joseph D, et al. P53 alteration and microsatellite instability have predictive value for survival benefit from chemotherapy in stage III colorectal carcinoma. Clinical cancer research : an official journal of the American Association for Cancer Research. 2001;7:1343-9.
[20] Ulrich CM. Nutrigenetics in cancer research--folate metabolism and colorectal cancer. J Nutr. 2005;135:2698-702.
[21] Zhao R, Matherly LH, Goldman ID. Membrane transporters and folate homeostasis: intestinal absorption and transport into systemic compartments and tissues. Expert Rev Mol Med. 2009;11:e4.
[22] Westerhof GR, Schornagel JH, Kathmann I, Jackman AL, Rosowsky A, Forsch RA, et al. Carrier- and receptor-mediated transport of folate antagonists targeting folate-dependent enzymes: correlates of molecular-structure and biological activity. Mol Pharmacol. 1995;48:459-71.
[23] Shayeghi M, Latunde-Dada GO, Oakhill JS, Laftah AH, Takeuchi K, Halliday N, et al. Identification of an intestinal heme transporter. Cell. 2005;122:789-801.
[24] Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature. 1998;391:806-11.
[25] Garofalo M, Quintavalle C, Di Leva G, Zanca C, Romano G, Taccioli C, et al. MicroRNA signatures of TRAIL resistance in human non-small cell lung cancer. Oncogene. 2008;27:3845-55.
[26] Giard DJ, Aaronson SA, Todaro GJ, Arnstein P, Kersey JH, Dosik H, et al. In vitro cultivation of human tumors: establishment of cell lines derived from a series of solid tumors. J Natl Cancer Inst. 1973;51:1417-23.
[27] Foster KA, Oster CG, Mayer MM, Avery ML, Audus KL. Characterization of the A549 cell line as a type II pulmonary epithelial cell model for drug metabolism. Experimental cell research. 1998;243:359-66.
[28] Tao SN, He XD, Shen ZJ, Dong L. [Differential invasion and metastasis capacities of methotrexate enantiomer-resistant A549 cell lines]. Zhonghua yi xue za zhi. 2012;92:2509-12.
[29] Feng H, Liu Q, Zhang N, Zheng L, Sang M, Feng J, et al. Leptin promotes metastasis by inducing an epithelial-mesenchymal transition in A549 lung cancer cells. Oncology research. 2014;21:165-71.
[30] Lieber M, Smith B, Szakal A, Nelson-Rees W, Todaro G. A continuous tumor-cell line from a human lung carcinoma with properties of type II alveolar epithelial cells. International journal of cancer Journal international du cancer. 1976;17:62-70.
[31] Thomson SP, Williams DB. Delineation of the lectin site of the molecular chaperone calreticulin. Cell stress & chaperones. 2005;10:242-51.
[32] Fliegel L, Burns K, MacLennan DH, Reithmeier RA, Michalak M. Molecular cloning of the high affinity calcium-binding protein (calreticulin) of skeletal muscle sarcoplasmic reticulum. The Journal of biological chemistry. 1989;264:21522-8.
[33] Coppolino MG, Woodside MJ, Demaurex N, Grinstein S, St-Arnaud R, Dedhar S. Calreticulin is essential for integrin-mediated calcium signalling and cell adhesion. Nature. 1997;386:843-7.
[34] Burns K, Duggan B, Atkinson EA, Famulski KS, Nemer M, Bleackley RC, et al. Modulation of gene expression by calreticulin binding to the glucocorticoid receptor. Nature. 1994;367:476-80.
[35] Lopez Sambrooks C, Carpio MA, Hallak ME. Arginylated calreticulin at plasma membrane increases susceptibility of cells to apoptosis. The Journal of biological chemistry. 2012;287:22043-54.
[36] Paquet ME, Leach MR, Williams DB. In vitro and in vivo assays to assess the functions of calnexin and calreticulin in ER protein folding and quality control. Methods. 2005;35:338-47.
[37] Bini L, Magi B, Marzocchi B, Arcuri F, Tripodi S, Cintorino M, et al. Protein expression profiles in human breast ductal carcinoma and histologically normal tissue. Electrophoresis. 1997;18:2832-41.
[38] Kageyama S, Isono T, Iwaki H, Wakabayashi Y, Okada Y, Kontani K, et al. Identification by proteomic analysis of calreticulin as a marker for bladder cancer and evaluation of the diagnostic accuracy of its detection in urine. Clinical chemistry. 2004;50:857-66.
[39] Toquet C, Jarry A, Bou-Hanna C, Bach K, Denis MG, Mosnier JF, et al. Altered Calreticulin expression in human colon cancer: maintenance of Calreticulin expression is associated with mucinous differentiation. Oncology reports. 2007;17:1101-7.
[40] Yoon GS, Lee H, Jung Y, Yu E, Moon HB, Song K, et al. Nuclear matrix of calreticulin in hepatocellular carcinoma. Cancer research. 2000;60:1117-20.
[41] Bergner A, Kellner J, Tufman A, Huber RM. Endoplasmic reticulum Ca2+-homeostasis is altered in Small and non-small Cell Lung Cancer cell lines. Journal of experimental & clinical cancer research : CR. 2009;28:25.
[42] Seliger B, Stoehr R, Handke D, Mueller A, Ferrone S, Wullich B, et al. Association of HLA class I antigen abnormalities with disease progression and early recurrence in prostate cancer. Cancer immunology, immunotherapy : CII. 2010;59:529-40.
[43] Zamanian M, Veerakumarasivam A, Abdullah S, Rosli R. Calreticulin and cancer. Pathology oncology research : POR. 2013;19:149-54.
[44] Du XL, Yang H, Liu SG, Luo ML, Hao JJ, Zhang Y, et al. Calreticulin promotes cell motility and enhances resistance to anoikis through STAT3-CTTN-Akt pathway in esophageal squamous cell carcinoma. Oncogene. 2009;28:3714-22.
[45] Frisch SM, Screaton RA. Anoikis mechanisms. Current opinion in cell biology. 2001;13:555-62.
[46] Melle C, Osterloh D, Ernst G, Schimmel B, Bleul A, von Eggeling F. Identification of proteins from colorectal cancer tissue by two-dimensional gel electrophoresis and SELDI mass spectrometry. International journal of molecular medicine. 2005;16:11-7.
[47] Wei DF, Wei YX, Cheng WD, Yan MF, Su G, Hu Y, et al. Proteomic analysis of the effect of triterpenes from Patrinia heterophylla on leukemia K562 cells. Journal of ethnopharmacology. 2012;144:576-83.
[48] Lo WY, Tsai MH, Tsai Y, Hua CH, Tsai FJ, Huang SY, et al. Identification of over-expressed proteins in oral squamous cell carcinoma (OSCC) patients by clinical proteomic analysis. Clinica chimica acta; international journal of clinical chemistry. 2007;376:101-7.
[49] Shalloe F, Elliott G, Ennis O, Mantle TJ. Evidence that biliverdin-IX beta reductase and flavin reductase are identical. The Biochemical journal. 1996;316 ( Pt 2):385-7.
[50] Singh SV, Iqbal J, Krishan A. Cytochrome P450 reductase, antioxidant enzymes and cellular resistance to doxorubicin. Biochemical pharmacology. 1990;40:385-7.
[51] Liu H, Rodgers ND, Jiao X, Kiledjian M. The scavenger mRNA decapping enzyme DcpS is a member of the HIT family of pyrophosphatases. The EMBO journal. 2002;21:4699-708.
[52] Kwasnicka-Crawford DA, Vincent SR. Role of a novel dual flavin reductase (NR1) and an associated histidine triad protein (DCS-1) in menadione-induced cytotoxicity. Biochemical and biophysical research communications. 2005;336:565-71.
[53] Crudden G, Chitti RE, Craven RJ. Hpr6 (heme-1 domain protein) regulates the susceptibility of cancer cells to chemotherapeutic drugs. The Journal of pharmacology and experimental therapeutics. 2006;316:448-55.
[54] Mifsud W, Bateman A. Membrane-bound progesterone receptors contain a cytochrome b5-like ligand-binding domain. Genome biology. 2002;3:RESEARCH0068.
[55] Crudden G, Loesel R, Craven RJ. Overexpression of the cytochrome p450 activator hpr6 (heme-1 domain protein/human progesterone receptor) in tumors. Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine. 2005;26:142-6.
[56] Neubauer H, Ruan X, Schneck H, Seeger H, Cahill MA, Liang Y, et al. Overexpression of progesterone receptor membrane component 1: possible mechanism for increased breast cancer risk with norethisterone in hormone therapy. Menopause. 2013;20:504-10.
[57] Difilippantonio S, Chen Y, Pietas A, Schluns K, Pacyna-Gengelbach M, Deutschmann N, et al. Gene expression profiles in human non-small and small-cell lung cancers. European journal of cancer. 2003;39:1936-47.
[58] Xu J, Zeng C, Chu W, Pan F, Rothfuss JM, Zhang F, et al. Identification of the PGRMC1 protein complex as the putative sigma-2 receptor binding site. Nature communications. 2011;2:380.
[59] Neubauer H, Adam G, Seeger H, Mueck AO, Solomayer E, Wallwiener D, et al. Membrane-initiated effects of progesterone on proliferation and activation of VEGF in breast cancer cells. Climacteric : the journal of the International Menopause Society. 2009;12:230-9.
[60] Yang T, Espenshade PJ, Wright ME, Yabe D, Gong Y, Aebersold R, et al. Crucial step in cholesterol homeostasis: sterols promote binding of SCAP to INSIG-1, a membrane protein that facilitates retention of SREBPs in ER. Cell. 2002;110:489-500.
[61] Rohe HJ, Ahmed IS, Twist KE, Craven RJ. PGRMC1 (progesterone receptor membrane component 1): a targetable protein with multiple functions in steroid signaling, P450 activation and drug binding. Pharmacology & therapeutics. 2009;121:14-9.
[62] Peluso JJ, Pappalardo A, Losel R, Wehling M. Progesterone membrane receptor component 1 expression in the immature rat ovary and its role in mediating progesterone's antiapoptotic action. Endocrinology. 2006;147:3133-40.
[63] Neubauer H, Clare SE, Wozny W, Schwall GP, Poznanovic S, Stegmann W, et al. Breast cancer proteomics reveals correlation between estrogen receptor status and differential phosphorylation of PGRMC1. Breast cancer research : BCR. 2008;10:R85.
[64] Zhang D, Putti TC. Over-expression of ERp29 attenuates doxorubicin-induced cell apoptosis through up-regulation of Hsp27 in breast cancer cells. Experimental cell research. 2010;316:3522-31.
[65] Gao D, Bambang IF, Putti TC, Lee YK, Richardson DR, Zhang D. ERp29 induces breast cancer cell growth arrest and survival through modulation of activation of p38 and upregulation of ER stress protein p58IPK. Laboratory investigation; a journal of technical methods and pathology. 2012;92:200-13.
[66] Concannon CG, Orrenius S, Samali A. Hsp27 inhibits cytochrome c-mediated caspase activation by sequestering both pro-caspase-3 and cytochrome c. Gene expression. 2001;9:195-201.
[67] Bareford MD, Park MA, Yacoub A, Hamed HA, Tang Y, Cruickshanks N, et al. Sorafenib enhances pemetrexed cytotoxicity through an autophagy-dependent mechanism in cancer cells. Cancer research. 2011;71:4955-67.
[68] Orrenius S, Zhivotovsky B, Nicotera P. Regulation of cell death: the calcium–apoptosis link. Nature Reviews Molecular Cell Biology. 2003;4:552-65.
[69] McGinnis KM, Gnegy ME, Park YH, Mukerjee N, Wang KK. Procaspase-3 and poly(ADP)ribose polymerase (PARP) are calpain substrates. Biochemical and biophysical research communications. 1999;263:94-9.
[70] Nakagawa T, Yuan J. Cross-talk between two cysteine protease families. Activation of caspase-12 by calpain in apoptosis. The Journal of cell biology. 2000;150:887-94.
[71] Reddy RK, Lu J, Lee AS. The endoplasmic reticulum chaperone glycoprotein GRP94 with Ca(2+)-binding and antiapoptotic properties is a novel proteolytic target of calpain during etoposide-induced apoptosis. The Journal of biological chemistry. 1999;274:28476-83.
[72] Bando Y, Katayama T, Aleshin AN, Manabe T, Tohyama M. GRP94 reduces cell death in SH-SY5Y cells perturbated calcium homeostasis. Apoptosis : an international journal on programmed cell death. 2004;9:501-8.
[73] Vazquez de la Torre A, Junyent F, Folch J, Pelegri C, Vilaplana J, Auladell C, et al. PI3 k/akt inhibition induces apoptosis through p38 activation in neurons. Pharmacological research : the official journal of the Italian Pharmacological Society. 2013;70:116-25.
[74] Lee J, Hong F, Kwon S, Kim SS, Kim DO, Kang HS, et al. Activation of p38 MAPK induces cell cycle arrest via inhibition of Raf/ERK pathway during muscle differentiation. Biochemical and biophysical research communications. 2002;298:765-71.
[75] Chuang SM, Wang IC, Yang JL. Roles of JNK, p38 and ERK mitogen-activated protein kinases in the growth inhibition and apoptosis induced by cadmium. Carcinogenesis. 2000;21:1423-32.
[76] Kralova J, Dvorak M, Koc M, Kral V. p38 MAPK plays an essential role in apoptosis induced by photoactivation of a novel ethylene glycol porphyrin derivative. Oncogene. 2008;27:3010-20.
[77] Cai B, Chang SH, Becker EB, Bonni A, Xia Z. p38 MAP kinase mediates apoptosis through phosphorylation of BimEL at Ser-65. The Journal of biological chemistry. 2006;281:25215-22.
[78] Ambrosino C, Nebreda AR. Cell cycle regulation by p38 MAP kinases. Biology of the cell / under the auspices of the European Cell Biology Organization. 2001;93:47-51.
[79] Kim BJ, Ryu SW, Song BJ. JNK- and p38 kinase-mediated phosphorylation of Bax leads to its activation and mitochondrial translocation and to apoptosis of human hepatoma HepG2 cells. The Journal of biological chemistry. 2006;281:21256-65.
[80] Xu RH, Pelicano H, Zhou Y, Carew JS, Feng L, Bhalla KN, et al. Inhibition of glycolysis in cancer cells: a novel strategy to overcome drug resistance associated with mitochondrial respiratory defect and hypoxia. Cancer research. 2005;65:613-21.
[81] Gottesman MM, Fojo T, Bates SE. Multidrug resistance in cancer: role of ATP-dependent transporters. Nature reviews Cancer. 2002;2:48-58.