摘要
目的：建立人肺腺癌耐药细胞模型Anip973／NVB，检测其生物学特性并从基因和蛋白质水平上探讨其诺维本的耐药机制。方法：本研究应用人肺腺癌细胞系Anip973，采用诺维本逐步增加剂量法，诱导建立人肺腺癌耐药细胞模型Anip973／NVB，检测其耐药细胞系和亲代细胞系的生物学特性；用基因芯片技术检测两细胞系间基因表达的差异；通过RT-PCR法验证部分基因芯片结果及检测传统耐药相关基因的表达；用2-DE技术初步分析其耐药细胞系与亲代细胞系间蛋白质表达的差异，探讨其诺维本的耐药机制。结果：1、本研究经过24个月建立了人肺腺癌耐药细胞系Anip973／NVB。2、通过Anip973／NVB生物学特性分析发现：(1)光镜及电子显微镜观察，Anip973／NVB细胞与Anip973细胞比较细胞形态不规则，呈长伪足的多角形及体积增大；线粒体数量增多，粗面内质网脱颗粒．(2)与亲代细胞相比Anip973／NVB细胞生长曲线的倍增时间无明显差异；(3)细胞周期分析发现：耐药细胞系Anip973／NVB的G_0-G_1期细胞明显增多，为62．90％，S期细胞明显减少，为28．32％，而亲代细胞系Anip973的上述各周期的百分比分别为53．73％、38．25％，两种细胞系的G_2-M期细胞无明显变化；(4)经MTT法测定Anip973／NVB细胞比较亲代细胞的NVB半数致死浓度(IC_(50))增大至21．81倍，该耐药细胞系同时对顺铂、紫杉醇、异环磷酰胺、表阿霉素、氟尿嘧啶、达卡巴嗪、足叶乙甙等药物也产生了交叉耐药，耐药倍数分别为11．89、11．68、6．12、3．95、2．86、2．64、2．15，而对伊立替康、吉西他滨相对敏感，耐药倍数分别为1．38、1．27(均P＜0．05=(5)高效液相色谱法测定发现，耐药细胞中的药物浓度明显低于亲代细胞。3、基因芯片结果表明，在检测的21571对基因中，Anip973／NVB和Anip973细胞均有差异表达的基因143对，其中在Anip973／NVB细胞中表达上调的55对，下调的88对。在这143对基因中，已知功能的基因有77条，明确与肿瘤相关的基因21对，在这些基因中ANXA1、ABCC3、TIMP-3和部分细胞粘连相关基因的表达发生了变化，这可能是导致诺维本耐药的主要机制；4、通过RT-PCR检测经典的耐药相关基因，结果显示：MRP3及Bcl-2基因在耐药细胞系中表达增高，而未发现MDR、MRP1、ABCG2、GSTp1基因在这两种细胞系中的差异表达。5、通过双向凝胶电泳(2-DE)技术，筛选两种细胞系表达差异的蛋白，获得了分辨率较高，重复性较好的2-DE图谱。在亲代细胞系中检测到的平均蛋白质点为806个，平均匹配率为77％；在耐药细胞系中检测到的平均蛋白质点为711个，平均匹配率为78．33％。筛选出在两种细胞系中表达差异的蛋白质37个，其中在耐药细胞中上调的蛋白16个，下调的蛋白21个。查询数据库初步鉴定了17个蛋白。结论：1、初步证明了Anip973／NVB细胞系是一个明确的多药耐药细胞模型，具有耐药细胞的基本生物学特性。2、基因芯片结果表明，Anip973／NVB的耐药机制可能是ANXA1、ABCC3基因表达增强导致细胞内药物浓度的降低；细胞浸润转移相关基因表达的下调，使肿瘤细胞分化趋向成熟，逃避了化疗药物的攻击；3、RT-PCR证明ABCC3(MRP3)在Anip973／NVB细胞系中表达增高，与本实验基因芯片结果符合，表明MRP3的过表达可能是Anip973／NVB细胞系耐药的主要机制之一；Bcl-2的过表达也参与了该细胞系的耐药；4、两种细胞系部分差异蛋白鉴定结果表明，趋化素样因子超家族2、NME2蛋白、细胞色素P450、金属基质蛋白酶抑制剂-4、层粘连蛋白受体、钙结合蛋白等蛋白质在两种细胞系中表达存在差异，说明诺维本在对人肺腺癌细胞的分化增殖等方面产生了影响，并提示这些蛋白质可能参与了Anip973／NVB细胞系的耐药。
Objective To establish a human lung adenocarcinoma multidrug-resistant cell lineAnip973/NVB and to study its biologic characteristics and mechanism of drugresistance.
Methods The human lung adenocarcinoma multidrug-resistant cell line(A973/NVB) was established by inducing human lung adenocarcinoma cell lineAnip973 with step-wise increasing concentrations of NVB,and the biologiccharacteristics were observed.cDNA microarray technique was used to detect genedifference of the two cell lines.The results of microarray were validated and theclassics drug resistance related genes were detected by RT-PCR.2-DE techniquewas used to analyze the difference of proteins of the two cell lines.
Results 1.The human lung adenocarcinoma multidrug-resistant cell lineAnip973/NVB was established successfully for 24 months.2.Its biologiccharacteristics were:(1) The cell morphous of A973/NVB cells were more irregularthan A973 cells,and there was significant difference in ultramicrostructure whileobserved by the light microscope and electron microscope.(2) The rate of cellproliferation of Anip973/NVB was no significantly different with that of Anip973cell line.(3) Cell cycle distribution of Anip973/NVB had changed compared withparental cells from flow cytometry.The percentage of cells in G_0-G_1 phase increasedto 62.90%,while the percentage of cells in S phase decreased to 28.32%.Thepercentage of Anip973 in G_0-G_1 and S phase were 53.73 %,38.25 % respectively,and there was no changes in G_2-M phase of the two cell lines.(4) The IC_(50) of NVB inAnip973/NVB cells was 21.18 times than that of Anip973 cells by MTT.In addition, the Anip973/NVB showed various cross-resistance to CDDP,isofosfamide,pharmorubicin,5-fluorouracil,dacarbazine and etoposide,and the resistance indexeswere 11.89,11.68,6.12,3.95,2.86,2.64 and 2.15,respectively.The ceils remainedsensitive to irinotecan and gemcitabine and the sensitive resistance indexes were 1.38and 1.27 (p＜0.05).(5)Medicine concentration in drug resistance cells was obviouslylower than that in parental cells by high efficiency liquid chromatography.3.The resultsof gene arrays showed that 143 genes of 21571 genes were differentially expressedbetween Anip973 and Anip973/NVB cell lines,of them,55 genes showed＞2 timesup-regulation,88 genes showed＜0.5 times down-regulation and the function ofseventy-seven genes had been identified.21 genes among identified genes had beenidentified function related to cancer,and,of 21 genes,the changes of ANXA1,ABCC3,TIMP-3 and cytoadherence related genes might be the major mechanism ofdrug resistance of NVB.4.The results by RT-PCR showed MRP3 and Bcl-2 wereoverexpressed in Anip973/NVB,and there were no significant expression of MDR,MRP1,ABCG2,GSTπ1 in two cell lines.5.Well-resolved,reproducible 2-DEpatterns of control and NVB-treated Anip973 were obtained.In Anip973/NVB group,average spots of 3 gels were 806 with an average matching rate of 77%,in Anip973group,average spots of 3 gels were 711 with an average matching rate of 78.33%.37significantly expressed protein spots were detected,among which,16 wereup-regulated and 21 were down-regulated in the drug-resistance group.17 spotswere identified initially with the data bank.
Conclusions 1.A973/NVB is a reliable multi-drug resistant cell line of humanlung adenocarcinoma which has the basic biologic characteristics.2.Up regulationof ANXA1 and ABCC3 result in depression of intra-cellular drug concentration andchange of intra-cellular drug distribution,which may be the major mechanism ofdrug resistance of NVB.Down regulation of cell infiltration and metastasis relatedgenes result in better of cell differentiation and elusion of attack fromchemotherapeutics.3.Overexpression of ABCC3(MRP3) may be one of the major mechanism of MDR,that is consistent with the results of gene arrays.Overexpression of Bcl-2 inhibits apoptosis of drug resistance cells which is one ofthe drug resistance cause.4.Difference of chemokine-like factor superfamily 2,NME2 protein,Cytochrome P450,TIMP-4,laminin receptor,Calumenin,and so on,in the two cells shows NVB affects lung adenocarcinoma cell differentiation andproliferation,and the proteins may play a role in NVB resistance.
引文
1 Bielder JL,Riehm H.Cellular resistance to actinomycin D in Chiness hamster cells in vitro:cross resistance,radioauto graphic and cytogenetic studies.Cancer Res,1970,30(4):1174-1179.
2 Uchiyama-kokubu N,Walanabe T.Establishment and characterization of adriamycin-resistant human colorectal adenocarcinoma HCT-15 cell lines with multidrug resistance.Anticancer Drugs,2001,12(9):769-778.
3 Hosking LK.Whelan RD.Shellard SA,et al.Multiple mechanisms of resistance in a series of human testicular teratoma cell lines selected for increasing resistance to etoposide:Int J Cancer,1994,57(2):259-237.
4 崔竹梅，向阳，杨秀玉．绒毛膜癌耐药细胞系的建立及人白细胞介素2基因转染后对其多耐药性的逆转作用．中华妇产科杂志，2001，36(9)：549-553．
5．陈杰，钱桂生，黄桂君．人肺腺癌多药耐药细胞系的建立及其生物学特征．第三军医大学报，2001，23(2)：135-139．
6 Oda Y,Matsumoto Y,Harimaya K,et al.Establishment of new multidrug-resistant human osteosarcoma cell lines.Oncol Rep,2000,7(4):859-866.
7 Kaye SB,Scotland G.Clinical drug resistance:The role of factors other than P-glycoprotein.Am J Med,1995,99(suppl 6A):40-44.
8 Shtil AA.Emergence of multidrug resistance in leukemia cells during chemotherapy:mechanisms and prevention.J Hematother Stem Cell Res,2002,11(2):231-41.
9 Mobus VJ,Gerharz CD,Weikel W,et al.Characterization of a human carcinosarcoma cell line of the ovary established after in vivo change of histologic differentiation.Gynecol Oncol,2001,83(3):523-532.
10 Yu DS,Ma CP,Chang SY,et al.Establishment and characterization of renal cell carcinoma cell lines with multidrug resistance.Urol Res,2000,28(2):86-92.
11 马胜林，冯建国，顾琳惠，凌雨田．耐药人肺癌细胞模型D6／MVP的建立及其生物学特性．中华肿瘤杂志，2002，25(2)：134-136．
12 陈建利，杨瑞芳，刘福生．卵巢上皮性癌顺铂耐药细胞株的建立及其多药耐药蛋白的表达．中华妇产科杂志，2000，35(10)：617-622．
13 蔡鹏，刘叙仪，韩复生，等．耐顺铂人肺腺癌细胞系A549DDP的建立及耐药机制．中国肿瘤临床，1995，22(8)：582-587．
14 Bowetel DD.Options available-from start to finish-for obtaining expression data by microarray[J].Nat Genet,1999,21(1 Suppl):25-32.
15 Kudoh K,Rarnanna M,Ravatn R,et al.Monitoring the expression profiles of doxorubicin-induced and doxorubicin-resistant cancer cels by cDNA microarray[J]Cancer Res,2000,60(15)4146-4166.
16 Komatani H,Kotani H,Hara Y,et al.Identification of breast cancer resistant protein/mitoxantrone Resistance/placenta-specific ATP-binding cassete transporter as a transporter of NB-506 and J-107088,topoisomerse I inhibitors with an indolocarbazole structure[J],Cancer Res,2001,61(7):2827-2832.
17 Hummerich L,Muller R,Hess J,et al.Identification of novel tumour-associated genes differentially expressed in the process of squamous cell cancer development.Oncogene.2006 Jan 5;25(1):111-21.
18 Oh P,Li Y,Yu J,et al.Subtractive proteomic mapping of the endothelial surface in lung and solid tumours for tissue-specific therapy[J],Nature,2004,429(6992):629-635.
19 Ahn SH,Sawada H,Ro JY,et al.Differential expression of annexin I in human mammary ductalepithelial cells in normal and benign and malignant breast tissues[J].Clin ExpM etastasis,1997,15(2):151-156.
20 De Coupade C,Gillet R,Bennoun M,et al.Annexin 1 expression and phosphorylation are upregulated during liver regeneration and transformation in antithrombin Ⅲ SV40 T large antigen transgenic mice[J].Hepatology,2000,31(2):371-380.
21 Bai XF, Ni XG, Zhao P, et al. Overexpression of annexin 1 in pan-creatic cancer and its clinical significance [J]. World J Gastroenterol, 2004, 10 (10):1466-1470.
22 Patel SK, Ma N, Monks TJ, et al. Changes in gene expression during chemical-induced nephrocarcinogenicity in the Eker rat[J]. Mol Carcinog, 2003,38 (3): 141-154.
23 Xia SH, Hu LP, Hu H, et al. Three isoforms of annexin I are preferentially expressed in normal esophageal epithelia but down-regulated in esophageal squamous cell carcinomas[J]. Oncogene, 2002, 21(43): 6641-6648.
24 Kang JS, Calvo BF, Maygarden SJ, et al. Dysregulation of annexin I protein expression in high-grade prostatic intraepithelial neoplasia and prostate cancer[J]. Clin Cancer Res, 2002, 8 (1): 117-123.
25 Vishwanatha JK, Salazar E, Gopalakrishnan VK. Absence of annexin I expression in B-cell non-Hodgkin's lymphomas and cell lines[J]. BMC Cancer,2004,4 (1): 1-8.
26 Garcia Pedrero JM, FernandezMP, Morgan RO, et al. Annexin A1 down-regulation in head and neck cancer is associated with epithelialdifferentiation status[ J ]. Am J Pathol, 2004,164 (1): 73-79.
27 Rosengaarth A, Gerke V, Lueche H. X-ray structure of full-length annexin 1 and implications for membrane aggregation [J]. J Mol Biol,2001, 306 (3):489-498.
28 Smitherman AB, Mohler JL, Maygarden SJ, et al. Expression of annexin I, Ⅱ and Ⅶ proteins in androgen stimulated and recurrent prostate cancer[J]. J U rol,2004,171 (2 Pt 1): 916-920.
29 Falini B, Tiacci E, Liso A, et al. Simple diagnostic assay for hairy cell leukaemia by immunocytochemical detection of annexin Al (ANXA1) [J]. Lancet, 2004, 363 (9424): 1869-1870.
30 Wu YL, Jiang XR, Lillington DM, et al. Upregulation of lipocortinl inhibits tumour necrosis factor-induced apoptosis in human leukaemic cells:a possible mechanism of resistance to immune surveillance[J].B r J Haem atol,2000,111(3):807-816.
31 Gehrmann ML,Fenselau C,Hathout Y.Highly altered protein expression profile in the adriamycin resistant MCF-7 cell line.J Proteome Res.2004;3(3):403-9.
32 Wang Y,Serfass L,Roy MO,et al.Annexin-I expression modulates drug resistance in tumor cells.Biochem Biophys Res Commun.2004,6;314(2):565-70.
33 左明新，叶爱军．多药耐药性相关ATP结合盒转运蛋白[J]．国外医学药学分册，2005，32(6)：365-368．
34 Bodo A,Bakos E,Szeri F,et al.The role of multidrug transporters in drug availability,metabolism and toxicity.Toxicol Lett.2003 Apr 11;140-141:133-43.
35 Wijnholds J,Drug resistance caused by multidrug resistance-associated proteins.Novartis Found Symp,2002,243:69-79.
36 Lai L,Tan TM.Role of glutathione in the multidrug resistance protein 4(MRP4/ABCC4)-mediated efflux of cAMP and resistance to purine analogues.Biochem J,2002,361(pt3):497-503.
37 Sampath J,Adachi M,Hatse S,et al.Role of MRP4 and MRP5 in biology chemotherapy.AAPS Pharm Sci,2004,4(3):E14.
38 Kruh G Zeng H,Rea PA,et al.MRP subfamily transporters and resistance to anticancer agents.J Bioenerg Biomember,2001,33(6):493-501.
39 Hsia Tc,Lin C,Wang J,et al.Relationship between chemotherapy response of small cell lung cancer and P-glycoprotein or multidrug resistance-related protein expression.Lung,2002,180(3):173-179.
40 Young LC,Campling BG,Cole SP,et al.Multidrug resistance proteins MRP3,MRP1 and MRP2 in lung cancer:correlation of protein levels with drug response and messenger RNA levels.Clin Cancer Res,2001,7(6):1798-1804.
41 Maata M, Soini Y, Liakka A, et al. Localization of MMP , TIMP-1, TIMP-2,TIMP-3 and TIMP-3 messenger RNA in normal, hyperplastic and neoplastic endometrium. Enhanced expression by endometrial adenocarcinomas is associated with low differentiation [J]. Am J Clin Pathol, 2000,114 (3): 402.
42 Kugler A, Matrix metalloproteinases and their inhibitors [J]. Anticancer Rse,1999,19(2c): 1589.
43 Mattila L, Airola K, Ahonen M, et al. Activation of tissue inhibitor of me-talloproteinases-3 (TIMP-3) mRNA expression in scleroderma skin fibrobla-sts.J Invest Dermatol, 1998,110: 416.
44 Fata J E, HO AT, Leco KJ, et al. Cellular turnover and extracellular matrix remodeling in femal reproductive tissue: function of MMPs and their inhi-bitors[J]. Cell Mol Life Sci, 2000,57(1): 77.
45 Kim M A, Lee H S, Yang H K, et al. Cytokeratin expression profile in gastric carcinomas[J]. Hum Pathol, 2004,35(5): 576-581.
46 Kimura K, Endo Y, Yonemura Y, et al. Clinical significance of S100A4 and E-cadherin-related adhesion molecules in non small cell lung cancer[J]. Int J Oncol, 2000,16(6): 1125-1131.
47 Platt-Higgins AM, Renshaw CA, West CR, et al. Comprison of the metastasis-inducing protein S100A4 (p9ka) with other prognostic markers in human breast cancer[J]. Int J Cancer, 2000,89(2): 198-208.
48 Davies BR, O'Donnell M, Durkan GC, et al. Expression of S100A4 protein is associated with metastasis and reduced survival in human bladder cancer[J]. J pathol, 2002,196(1): 292-299.
49 Shebet GV, Lakshmi MS. S100A4 (MTS1) calcium binding protein in cancer growth , invasion and metastasis [J] . Anticancer Res , 1998 , 18 (4A) :2415 - 2421.
50 Yonemura Y, Endou Y, Kimura K, et al . Inverse expression of S100A4 and E-cadherin is associated with metastatic potential in gastric cancer [J] . Clin Cancer Res,2001,92(6):832-838.
51 王蔚，孙青，韩金祥，等．用基因芯研究人肺癌转移相关基因[J]．J Clin Exp Pathol 2005 Apr，21(2)：155-163．
52 Kim M A,Lee H S,Yang H K,et al.Cytokeratin expression profile in gastric carcinomas[J].Hum Pathol,2004,35(5):576-581.
53 Luo A,Kong J,Hu G,et al.Discovery of Ca~(2+)-relevant and differentiationassociated genes downregulated in esophageal squamous cell carcinoma using eDNA microarray[J].Oncogene,2004,23(6):1291-1299.
54 Van de Rijn M,Perou C M,Tibshirani R,et al.Expression of cytokeratins 17 and 5 identifies a group of breast carcinomas with poor clinical outcome[J].American Journal of Pathology,2002,16(1):1991-1996.
55 Young LC,Campling BG,Voskoglou-Nomikos T,et al.Expression of multidrug resistance protein-related genes in lung cancer:correlation with drug response[J].Clin Cancer Res,1999,5[3]:673-680.
56 Qka M,Fukuda M,Sakamoto A,et al.The clinical role of MDR1 gene expression in human lung cancer[J].Anticancer Res,1997,179(1B):721-724.
57 Zhu L,Lang JH.P-glycoprotein and multidrug resistance[J].Chin J Obstet Gynecol,1998,33(66):381-383.
58 Glavinas H,Krajcsi P,Cserepes J,et al.The role of ABC transporters in drug resistance,metabolism and toxicity.Curr Drug Deliv.2004;1(1):27-42.
59 Torky AR,Stehfest E,Viehweger K,et al.Immuno-histochemical detection of MRPs in human lung cells in culture.Toxicology.2005 Feb 28;207(3):437-50.
60 Bodo A,Bakos E,Szeri F,et al.The role of multidrug transporters in drug availability,metabolism and toxicity.Toxicol Lett.2003 11;140-141:133-43.
61 Yoh K,Ishii G,Yokose T,et al.Breast cancer resistance protein impacts clinical outcome in platinum-based chemotherapy for advanced non-small cell lung cancer.Clin Cancer Res.2004 Mar 1;10(5):1691-7.
62 Hsia TC,Lin CC,Wang JJ,et al.Relationship between chemotherapy response of small cell lung cancer and P-glycoprotein or multidrug resistance-related protein expression. Lung. 2002;180(3):173-9.
63 Young LC, Campling BG, Cole SP, et al. Multidrug resistance proteins MRP3,MRP1, and MRP2 in lung cancer: correlation of protein levels with drug response and messenger RNA levels. Clin Cancer Res. 2001; 7(6): 1798-1804.
64 Kawai H, Kiura K, Tabata M, et al. Characterization of non-small-cell lung cancer cell lines established before and after chemotherapy. Lung Cancer.2002;35 (3):305-14.
65 Kim R, Emi M, Tanabe K, et al. Therapeutic potential of antisense Bcl-2 as a chemosensitizer for cancer therapy. Cancer. 2004 , l;101(11):2491-502.
66 Chen SY, Liu SX, Li XM. Expression of bcl-2 and bax in arsenic sulfide induced-apoptosis of K562 cell [J]. J Fourth Mil Med Univ. 2002,23(9):790-792
67 Wang J, Liu X, Jiang W.Co-transfection of MRP and bcl-2 antisense S-oligodeoxynucleotides reduces drug resistance in cisplatin-resistant lung cancer cells. Chin Med J (Engl). 2000 ; 113(10): 957-960.
68 Shiozawa K ,Oka M,Soda H, et al.Reversal of breast cancer resistance protein (BCRP/ABCG2)-mediated drug resistance by novobiocin,a coumermycin antibiotic.InJ Cancer,2004,108(1):146
69 Bessho Y, Oguri T, Achiwa H, et al. Role of ABCG2 as a biomarker for predicting resistance to CPT-11/SN-38 in lung cancer. Cancer Sci. 2006 Mar;97(3):192-8.
70 Elkind NB, Szentpetery Z, Apati A, et al. Multidrug transporter ABCG2 prevents tumor cell death induced by the epidermal growth factor receptor inhibitor Iressa (ZD1839, Gefitinib). Cancer Res. 2005 Mar l;65(5):1770-7.
71 Bai F, Nakanishi Y, Kawasaki M, et al. Immunohistochemical expression of glutathione S-transferase-Pi can predict chemotherapy response in patients with nonsmall cell lung carcinoma 〔J〕 . Cancer, 1996,78(3): 416.
72 Tew KD. TLK-286: a novel glutathione S-transferase-activated prodrug. Expert Opin Investig Drugs. 2005 Aug; 14(8):1047-54.
73 Turella P, Cerella C, Filomeni G, et al. Proapoptotic activity of new glutathione S-transferase inhibitors. Cancer Res. 2005 May 1;65(9):3751-61.
74 Corbett JM, Dunn MJ, Posch A, et al. Positional reproducibility of protein spots in two-dimensional polyacrylamide gel electrophoresis using immobilized pH gradient isoelectric focusing in the first dimension: an interlaboratory comparision[J]. Electrophoresis, 1994,15(8-9):1205-1211
75 O'Farrell PH . High resolution two- dimensional electrophoresis of proteins . J Biol Chem . 1975,250(10): 4007-4021
76 Klose J, Kobalz U. Two dimensional electrophoresis of proteins, an up-dated protocol and implications for a function analysis of the genome . Electrophosis .1995,16:1034.
77 Molloy MP. Two dimensional electrophoresis of membrane proteins using immobilized pH gradients. Anal Biochem, 2000,280(1):1-10
78 Fey SJ, Larsen PM. 2D or not 2D. Two-dimensional gel electrophoresis. Curr Opin Chem Biol. 2001 ;5(1):26-33.
79 Klein E, Klein JB, Thongboonkerd V. Two-dimensional gel electrophoresis: a fundamental tool for expression proteomics studies. Contrib Nephrol.2004;141:25-39.
80 Lim MS, Elenitoba-Johnson KS. Proteomics in pathology research. Lab Invest. 2004 ;84(10):1227-44.
81 Ferguson PL, Smith RD. Proteome analysis by mass spectrometry. Annu Rev Biophys Biomol Struct. 2003;32:399-424.
82 Gevaert K, Vandekerckhove J. Protein identification methods in proteomics.Electrophoresis. 2000 ;21(6):1145-54.
83 Yang YX, Xiao ZQ, Chen ZC, et al. Proteome analysis of multidrug resistance in vincristine-resistant human gastric cancer cell line SGC7901/VCR. Proteomics.2006;6(6):2009-21.
84 Chen CY, Jia JH, Zhang MX,et al. Proteomic analysis on multi-drug resistant cells HL-60/DOX of acute myeloblastic leukemia. Chin J Physiol. 2005, 48 (3):115-120.
85 Besada V, Diaz M, Becker M, et al. Proteomics of xenografted human breast cancer indicates novel targets related to tamoxifen resistance. Proteomics.2006;6(3):1038-1048.
86 Yoo BC, Ku JL, Hong SH, et al. Decreased pyruvate kinase M2 activity linked to cisplatin resistance in human gastric carcinoma cell lines. Int J Cancer. 2004,10;108(4):532-539.
87 Han WL, Lou YX, Tang JM, et al . Molecular cloning and characterization of chemokine-like factor 1(CKLF1) a novel human cytokine with unique structure and potencial chemotactic activity[J]. Biochem J,2001,357:127-135.
88 Shi S, Rui M, Han W, et al .CKLFSF2 is highly expressed in testis and can be secreted into the seminiferous tubules. Int J Biochem Cell Biol. 2005 Aug;37(8):1633-1640.
89 Li J, Zhou J, Chen G, et al . Inhibition of ovarian cancer metastasis by adeno-associated virus-mediated gene transfer of nm23Hl in an orthotopic implantation model. Cancer Gene Then 2006 Mar;13(3):266-272.
90 Jin S, Pan X, Wang Y. Effect of nm23Hl on proliferation, tumor formation and metastasis of hepatocarcinoma. Zhonghua Zhong Liu Za Zhi. 2000 Sep;22 (5):381-384.
91 Postel EH. Multiple biochemical activities of NM23/NDP kinase in gene regulation. J Bioenerg Biomembr. 2003 Feb;35(1):31-40.
92 Kimura N, Shimada N, Fukuda M,et al. Regulation of cellular functions by nucleoside diphosphate kinases in mammals. J Bioenerg Biomembr. 2000 Jun;32(3):309-315.
93 Elizabeth A. Kidd, Jinsheng Yu, Xia Li, et al. Variance in the Expression of 5-Fluorouracil Pathway Genes in Colorectal Cancer . Clinical Cancer Research. 2005(11): 2612-2619.
94 邱星辉,冷欣夫. 细胞色素 P450 与癌症基因治疗.生命的化学 .2000,(5):238-239
95 Visse R, Hideaki N. Matrix metalloproteinases and tissue inhibitors of metalloproteinases structure, function, and biochemistry [J].Cire Res, 2003,92:827
96 Lizarraga F, Espinosa M, Maldonado V,et al. Tissue inhibitor of metalloproteinases-4 is expressed in cervical cancer patients. Anticancer Res. 2005 Jan-Feb;25(1B):623-627.
97 Koyama S. Enhanced cell surface expression of matrix metalloproteinases and their inhibitors, and tumor-induced host response in progression of human gastric carcinoma. Dig Dis Sci. 2004 Oct;49(10):1621-1630.
98 Jiang X, Huang X, Li J,et al. Relationship between tissue inhibitors of metalloproteinase and metastasis and prognosis in breast cancer. Zhonghua Wai Ke Za Zhi. 2000 Apr;38(4):291-3,19.
99 Tunuguntla R, Ripley D, Sang QX, et al. Expression of matrix metalloproteinase-26 and tissue inhibitors of metalloproteinases TIMP-3 and -4 in benign endometrium and endometrial cancer. Gynecol Oncol. 2003 Jun;89(3):453-459.
100 Mannello F, Gazzanelli G. Tissue inhibitors of metalloproteinases and programmed cell death: conundrums, controversies and potential implications.Apoptosis. 2001 Dec;6(6):479-482.
101 Appierto V, Cavadini E, Pergolizzi R, et al. Decrease in drug accumulation and in tumour aggressiveness marker expression in a fenretinide-induced resistant ovarian tumour cell line. Br J Cancer. 2001 Jun 1;84(11):1528-1534.
102 Honore B, Vorum H. The CREC family, a novel family of multiple EF-hand,low-affinity Ca(2+)-binding proteins localised to the secretory pathway of mammalian cells. FEBS Lett. 2000 Jan 21;466(1):11-18.
103 Ding SJ, Li Y, Shao XX, et al. Proteome analysis of hepatocellular carcinoma cell strains, MHCC97-H and MHCC97-L, with different metastasis potentials.Proteomics. 2004 Apr;4(4):982-994.
1.Rhoads CD.Nitrogen mustards in treatment of neoplastic disease.JAMA,1946,21:656-677.
2.Bielder JL,Riehm H.Cellular resistance to actinomycin D in Chiness hamster cells in vitro:cross resistance,radioauto graphic and cytogenetic studies.Cancer Res,1970,30(4):1174-1179.
3.Shitil AA.Emergence of multidrug resistance in leukemia cells during chemotherapy:mechanisms and prevention.J Hematother Stem Cell Res,2002,11(2):231-239.
4．周向东，钱桂生，刘凌志．人小细胞肺癌SH77多药耐药细胞系的建立及其生物学特征分析．第三军医大学报，2003，25(16)：1405-1407．
5.Uchiyama-kokubu N,Walanabe T.Establishment and characterization of adriamycin-resistant human colorectal adenocarcinoma HCT-15 cell lines with multidrug resistance.Anticancer Drugs,2001,12(9):769-778.
6.Hosking LK.Whelan RD.Shellard SA,et al.Multiple mechanisms of resistance in a series of human testicular teratoma cell lines selected for increasing resistance to etoposide.Int J Cancer,1994,57(2):259-237.
7．崔竹梅，向阳，杨秀玉．绒毛膜癌耐药细胞系的建立及人白细胞介素2基因转染后对其多耐药性的逆转作用．中华妇产科杂志，2001，36(9)：549-553．
8．陈杰，钱桂生，黄桂君．人肺腺癌多药耐药细胞系的建立及其生物学特征．第三军医大学报，2001，23(2)：135-139．
9.Sandal T,Aahlgren R,Lillehaug J,et al.Establishment of okadaic acid resistant cell clones using a cDNA expression library.Cell Death Differ,2001,8(7):754-764.
10．刘友华．人白血病HL-60细胞系HGPRT缺失突变型细胞株的分离和建立．见：章静波，主编．细胞生物学实用方法与技术．第1版．北京：北京医科大学中国协和医科大学联合出版社，1995，62-68．
11．杨志林，徐如祥．三维细胞培养在肿瘤研究中的进展．国外医学肿瘤学分册，2001，28(4)：100-104．
12.Maurer BJ,Ihnat MA,Morgan C,et al.Growth of human tumor cells in macroporous relative to monolayers.Mol Pharmacol,1999,55(5):938-944.
1 Kool M,van der Linden M,de Haas M,et al.MRP3,an organic anion transporter able to transport anti-cancer drugs[J].Proc Natl Acad Sci USA,1999,96[12]:6914-6919.
2 Paredes-Lario A,Blanco-Garcia C,Echenique-Elizondo M.Expression of multiple-drug resistant proteins in lung cancer。 Cir Esp.2006 Jan;79(1):46-56.
3 Young LC,Campling BG,Voskoglou-Nomikos T,et al.Expression of multidrug resistance protein-related genes in lung cancer:correlation with drug response[J].Clin Cancer Res,1999,5[3]:673-680.
4 Qka M,Fukuda M,Sakamoto A,et al.The clinical role of MDR1 gene expression in human lung cancer 〔J〕. Anticancer Res, 1997,179(1B): 721-724.
5 Zhu L, Lang JH. P-glycoprotein and multidrug resistance 〔J〕 . Chin J Obstet Gynecol, 1998, 33(66): 381-383.
6 Torky AR, Stehfest E, Viehweger K, et al. Immuno-histochemical detection of MRPs in human lung cells in culture. Toxicology. 2005 Feb 28;207(3):437-50.
7 Cole SP, Deeley RG Multidrug resistance mediated by the ATP-binding cassette transporter protein MRP 〔J〕 . Bioessays, 1998, 20(ll):931-940.
8 Yoh K, Ishii G, Yokose T,et al. Breast cancer resistance protein impacts clinical outcome in platinum-based chemotherapy for advanced non-small cell lung cancer. Clin Cancer Res. 2004 Mar l;10(5):1691-7.
9 Oshika Y , Nakamura M, Tokunaga T, et al. Multidrug resistance-associated protein and mutant p53 protein expression in non-small-cell lung cancer 〔J〕 . Mod Pathol, 1998, 11(11):1059-1063.
10 Hsia TC, Lin CC, Wang JJ, et al. Relationship between chemotherapy response of small cell lung cancer and P-glycoprotein or multidrug resistance-related protein expression. Lung. 2002;180(3):173-9.
11 Kawabata S, Oka M, Shiozawa K, et al. Breast cancer resistance protein directly confers SN-38 resistance of lung cancer cells. Biochem Biophys Res Commun,2001, 280:1216-1223.
12 Meschini S, Marra M, Calcabrini A, et al. Role of the lung resistance-related protein (LRP) in the drug sensitivity of cultured tumor cells. Toxicol In Vitro.2002 Aug;16(4):389-98.
13 Sandri MI, Hochhauser D, Ayton P, et al. Differential expression of the topoisomerase Ⅱ α and β genes in human breast cancers. Br J Cancer,1996, 73:1518-1524.
14 FILIPITS M, STRANZL T, POHL G, et al. Clinical relevance of the lung resistance protein in diffuse large B- cell lymphomas 〔J〕 . Drug Clin Cancer Res, 2000, 6: 3417-3423.
15 Berger W,Elbling L,Micksche M.Expression of the major vault protein LRP in human non-small-cell lung cancer cells:activation by short-term exposure to antineoplastic drugs[J].Int J Cancer,2000,88(2):293-300.
16 Shiozawa K,Oka M,Soda H,et al.Reversal of breast cancer resistance protein(BCRP/ABCG2)-mediated drug resistance by novobiocin,a coumermycin antibiotic.InJ Cancer,2004,108(1):146.
17 Bessho Y,Oguri T,Achiwa H,et al.Role of ABCG2 as a biomarker for predicting resistance to CPT-11/SN-38 in lung cancer.Cancer Sci.2006 Mar;97(3):192-8.
18 Elkind NB,Szentpetery Z,Apati A,et al.Multidrug transporter ABCG2 prevents tumor cell death induced by the epidermal growth factor receptor inhibitor Iressa(ZD1839,Gefitinib).Cancer Res.2005 Mar 1;65(5):1770-7.
19 Bai F,Nakanishi Y,Kawasaki M,et al.Immunohistochemical expression of glutathione S-transferase-Pi can predict chemotherapy response in patients with nonsmall cell lung carcinoma[J].Cancer,1996,78(3):416.
20 Tew KD.TLK-286:a novel glutathione S-transferase-activated prodrug.Expert Opin Investig Drugs.2005 Aug;14(8):1047-54.
21 Turella P,Cerella C,Filomeni G,et al.Proapoptotic activity of new glutathione S-transferase inhibitors.Cancer Res.2005 May 1;65(9):3751-61.
22 王彦，吴焕明．肺癌中金属硫蛋白的表达及其与细胞增殖、凋亡的关系．中国组织化学与细胞化学杂志，2003，12(4)：393-398．
23 Rosell R,Danenberg K,Gandara D,et al.Altered ERCC1 expression correlates with response to cisplatin(DDP)+gemcitabine(GEM)in non-small-cell lung cancer(NSCLC)patients(pts).Proc Am Soc Clin Oncol,2001,20:326a.
24 Lord RV,Brabender J,Gandara D,et al.Low ERCCI expression correlates with prolonged survival after cisplatin plus gemcitabine chemotherapy in non-small cell lung cancer.Clin Cancer Res,2002,8(7):2286-2291.
25 Heim MM,Eberhardt W,Seeber S,et al.Differential modulation of chemosensitivity to alkylating agents and platinum compounds by DNA repair modulators in human lung cancer cell lines. J Cancer Res Clin Oncol,2000,126(4): 198-204.
26 Rusch V, Klimstra D, Venkatraman E, et al. Aberrant p53 expression predicts clinical resistance to cisplatin-based chemotherapy in locally advanced non-small cell lung cancer 〔J〕 . Cancer Res, 1995, 55(21): 5038-5042.
27 Kawasaki, Nakanishi Y, Kuwano K, et al. The utility of p53 immunostaining of tranosbronchial biopsy specimens in advanced non-small-cell lung cancer (J) .Clin Cancer Res, 1997, 3(7): 1195-1200.
28 Wang J, Liu X, Jiang W. Co-transfection of MRP and bcl-2 antisense S-oligodeoxynucleotides reduces drug resistance in cisplatin-resistant lung cancer cells. Chin Med J (Engl). 2000 Oct;113(10):957-60.
29 Nassif NT, Lobo GP, Wu X, et al. PTEN mutations are common in sporadic microsatellite stable colorectal cancer. Oncogene,2004, 23(2): 617-628.
30 Saga Y, Mizukami H, Suzuki M, et al. Overexpression of PTEN increases sensitivity to SN-38, an active metabolite of the topoisomerase I inhibitor irinotecan, in ovarian cancer cells. Clin Cancer Res, 2002, 8(5): 1248-1252.
31 Soria JC, Lee HY, Lee JI, et al. Lack of PTEN expression in non-small cell lung cancer could be related to promoter methylation. Clin Cancer Res, 2002, 8(5):1178-1184.
32 Falleni M, Pellegrini C, Marchetti A, et al. Survivin gene expression in early-stage non-small cell lung cancer. J Pathol, 2003,200 (5): 620-626.
33 Olie RA, Simoes-Wust AP, Baumann B, et al. A novel antisense oligonucleotide targeting survivin expression induces apoptosis and sensitizes lung cancer cells to chemotherapy 〔J〕 . Cancer Res, 2000, 60 (11): 2805-2809.
34 Verdier Pinard P, Wang F, Martello L, et al. Analysis of tubulin isotypes and mutations from taxol-resistant cells by combined isoelectrofocusing and mass spectrometry. Biochemistry, 2003, 42(18): 5349-5357.
35 ari M, Yang H, Zeng, C, et al. Expression of cless Ⅲ beta-tubulin reduces microtubule assembly and confers resistance to paclitaxel. Cell Motil Cytoskeleton, 2003,56(1): 45-56.
36 osell R, Scagliotti G, Danenberg KD, et al. Transcripts in pretreatment biopsies from a three-arm randomized trail in metastatic non-small-cell lung cancer.Oncogene, 2003,22(23): 3548-3553.