摘要
目的：通过体外实验观察沙利度胺对人乳腺癌细胞血管内皮生长因子-C(vascular endothelial growth factor-C，VEGF-C)及其受体(vascular endothelial growth factor receptor-2，VEGFR-2)的表达影响，进一步阐明沙利度胺抗血管生成作用的可能机制，探讨其用于治疗乳腺癌的可行性。
方法：选取两株经典的人乳腺癌细胞系MCF-7及MDA-MB-231，采用四甲基偶氮唑蓝(MTT)法检测沙利度胺不同浓度组(6、30、60、120μg/ml)、不同时间组(24、48、72h)对细胞存活和生长的影响情况，从而选择适当的药物浓度及作用时间。根据MTT的结果，设置对照及实验组。分别提取各组细胞总RNA，鉴定其完整性及含量，通过逆转录—聚合酶链反应(revers transcription PCR，RT-PCR)半定量检测沙利度胺处理前后两株细胞中VEGF-C、KDR mRNA的表达水平。应用免疫细胞化学(immunocytochemistry，IC)方法定性观察沙利度胺处理前后MCF-7、MDA-MB-231细胞中VEGF-C、KDR蛋白的表达情况。采用流式细胞术(flow cytometry，FCM)半定量检测沙利度胺处理前后两株细胞中VEGF-C及KDR蛋白的表达水平。
结果：1 MTT结果显示：沙利度胺在(6～120)μg/ml浓度范围内能明显抑制MCF-7和MDA-MB-231细胞体外增
Objective: This experiment aimed to evaluate the effect of thalidomide on the expression of vascular endothelial growth factor-C (VEGF-C) and its receptor (vascular endothelial growth factor receptor-2, VEGFR-2) in breast cancer cell lines in vitro to further investigate possible mechanism of its role as angiogenesis inhibitor and feasibility to cure breast cancer.
Methods: Selecting two-stub classic human breast cancer cell lines, MCF-7 and MDA-MB-231, using MTT assay to detect the growth rate among different thalidomide concentration groups (6、30、60、120μg/ml)and different time groups (24、48、72h), in order to choose proper drug concentration and action time. According to the result of MTT, establishing control and experimental group, extracting total RNA of each group cell, assessing the integrality and content of RNA, the level of VEGF-C、KDRmRNA expression was examined by semiquantitative RT-PCR technique in the two-stub cell lines treated before and after with thalidomide. The expression of VEGF-C、KDR protein was qualitationly studied by immunocytochemistry staining and was semi-quantitately examined by flow cytometry in cell lines treated before and after with thalidomide.
引文
1 易文君，唐中华，陈干农，等．乳腺癌VEGF，bFGF及其受体的表达和与微血管计数的关系[J]．中国现代医学杂志，2003，13(20)：39～13
2 Kaushal V, Mukunyadzi P, Dennis R A, et al. Stage-Specific Characterization of the Vascular Endothelial Growth Factor Axis in Prostate Cancer: Expression of Lymphangiogenic Markers is Associated with Advanced-Stage Disease [J]. Clin Cancer Res, 2005, 11:584～593
3 Al-Mowallad AF, Li C, Wilson P, et al. Quantification of vascular endothelial growth factor-C [J]. Jimmunol Methods, 2004, 289(1-2):239～240
4 Byrne A M, Bouchier-Hayes D J, Harmey J H. Angiogenic and cell survival functions of Vascular Endothelial Growth Factor (VEGF) [J]. J. Cell. Mol. Med, 2005, 9(4):777～794
5 Podar K, Anderson K C. The pathophysiologic role of VEGF in hematologic malignancies: therapeutic implications [J]. Blood, 2005, 105:383～1395
6 Bisacchi D, Benelli R, Vanzetto C et al. Anti-angiogensis and angioprevention mechanisms, problems and perspectives [J]. Cancer Detection and Prevention, 2003, 27(3):229～38
7 Reist M, Carrupt PA, Francotte E, et al. Chiral inversion and hydolysis of thalidomide: Mechanisms and catalysis by bases and serum albumin, and chiral stability of teratogenic metabolites [J]. Chem Res Toxicol, 1998, 11 (12): 1521～ 1528
8 Eriksson T, Bjorkman S, Roth B, et al. Intravenous formoul-ation of the enantiomers of thalidomide: pharmacolkinetic and initial pharmacodynamic characteriz- ation in man [J]. J Pharm Pharmacol, 2000, 52 (7):807～817
9 Musto P. Thalidomide thetapy for myelidysplastic syndromes: current status and future perspectived [J]. Leukemia Research, 2004, 28(4):325～332
10 Arrieta O, Guevara P, Tamariz J, et al. Antiproliferative effect of thalidomide alone and combined with carmustine against C6 rat glioma [J]. Int J Exp Pathol, 2002, 83(2):99～104
11 季艳霞，姜达，康振桥．沙利度胺对人乳腺癌细胞增殖及血管内皮生长因子表达的影响[J]．肿瘤学杂志，2005，11(1)：26～28
12 Joukov V, Pajusolak, Kaipainen A, et al. A novel vascular endothelial growth factor, VEGF-C is a ligand for the Flt-4 (VEGFR-3) and KDR (VEGFR-2) receptor tyrosine kinases [J]. The EMBO Journal, 1996, 15(2):290～298
13 Wissmann C, Hocker M, et al. VEGF-C, VEGF-D and VEGF-receptor3: novel key regulators of lymphangiogenesis and cancer metastasis [J]. Z Gastroenterol, 2002, 40(9):853～856
14 Li X, Eriksson U, et al. Novel VEGF family members: VEGF-B, VEGF-C and VEGF-D [J]. Int J Biochem Cell Biol, 2001, 33:421～426
15 Salven P, Lymboussaki A, Heikkila P, et al. Vascular endothelial growth factors VEGF-B and VEGF-C are expressed in human tumors [J]. Am J Psthol, 1998, 153(1): 103～108
16 高杰，刘执玉，毕玉顺等．血管内皮生长因子C mRNA人乳腺癌细胞株中的表达[J]．山东大学医学报(医学)版，2002，40(3)：219～221
17 Mattila MM, Ruohola JK, Karpanen T, et al. VEGF-C induced lymphangiogenesis is associated with lymph node metastasis in orthotopic MCF-7 tumors. VEGF-C induced lymphangiogenesis is associated with lymph node metastasis in orthotopic MCF-7 tumors [J]. Int J Cancer, 2002, 98(6): 946～951
18 Hamada K, Olike Y, Takakura N, et al. VEGF-C signaling pathways through VEGFR-2 and VEGFR-3 in vasculo-angiogenesis and hematopoiesis [J]. Blood, 2000, 96(12): 3793～3800
19 Makinen T, Veikkola T, Mustjoki S, et al. Isolated lymphatic endothelial cells transduce growth, survival and migratory signals via the VEGF-C/D receptor VEGFR-3 [J]. EMBO J, 2001, 20(17):4762～4773
20 Hiltunent MO, Laitinen M, Turunen MP, et al. Intravascular adenovirus-mediated VEGF-C gene transfer reduces neointima formation in balloon-denuded rabbit aorta [J]. Circulation, 2000, 102(18):2262～2268
21 Mimura T, Amano S, Usui T, et al. Expression of vascular endothelial growth factor C and vascular endothelial growth factor receptor 3 in corneal lymphangiogenesis [J]. Exp Eye Res, 2001, 72(1):71～78
22 Stacker SA, Williams RA, Achen MG. Lymphangiogenic growth factors as markers of tumor metastasis [J]. APMIS, 2004, 112(7-8):539～549
23 Tamura M, Ohta Y. Serum vascular endothelial growth factor-C level in patients with primary nonsmall cell lung carcinoma: a possible diagnostic tool for lymph node metastasie [J]. Cancer, 2003, 98(6): 1217～1222
24 Maeda K, Yashiro M, Nishihara T, et al. Correlation between vascular endothelial growth factor C expression and lymph node metastasis in t1 carcinoma of the colon and rectum [J]. Surg Today, 2003, 33(10):736～739
25 Lee TH, Avraham HK, Jiang S, et al. Vascular endothelial growth factor modulates the transendothelial migration of MDA-MB-231 breast cancer cells through regulation of brain microvascular endothelial cell permeability [J].J Biol Chem, 2003, 278(7):5277～5284
26 Gunningham SP, Currie MJ, Han C, et al. The short form of the alternatively spliced flt-4 but not its ligand vascular endothelial growth factor C is related to lymph node metastasis in human breast cancers [J]. Clin Cancer Res, 2000, 6:4278～4286
27 Neufeld G, Cohen T. Vascular endothelial growth factor (VEGF) and its receptors [J]. FASEB J, 1999, 13:9～22
28 Veikkola T, Karkkainen M, Claesson-Welsh L, et al. Regulation of angiogensis via vascular endothelial growth factor receptors [J]. Cancer Res, 2000, 60(2):203～212
29 Soker S, Takashima S, Miao H Q, et al. Neuropilin-1 is expressed by endothelial and tumor cells as an isoform-specific receptor for vascular endothelial growth factor [J]. Cell, 1998,92(6):735～745
30 Gerber H P, McMurtrey A, Kowalski J, et al. Vascular endothelial growth factor regulates endothelial cell survival through the phosphatidylinositol3'-kinase/Akt signal transduction pathway. Requirement for Flk-1/KDR activation [J]. J Biol Chem, 1998, 273(46):30336～30343
31 Brekken RA, Overholser JP, Stastny VA, et al. Selective inhibition of vascular endothelial growth factor (VEGF) receptor2 (KDR/Flk-1) activity by a monoclonal anti- VEGF antibody blocks tumor growth in mice [J]. Cancer Res, 2000, 60(18):5117～5124
32 Millauer B, Longhi M P, Plate K H, et al. Dominant-negative inhibition of Flk-1 suppresses the growth of many tumor types in vivo [J]. Cancer Res, 1996, 56(7):1615～1620
33 Masood R, Cai J, Zheng T, et al. Vascular endothelial growth factor (VEGF) is an autocrine growth factor for VEGF recptorpositiv human tumors [J]. Blood, 2001, 98(6):1904～1913
34 刘芳，张雅洁．VEGF-C及其受体Flt-4在乳腺癌细胞增殖及转移中的作用 [J]．癌症，2003，22(10)：1053～1056
35 Gunningham SP, Currie MJ, Han C, et al. The short form of the alternatively spliced flt-4 but not its ligand vascular endothelial growth factor C is related to lymph node metastasis in human breast cancers [J]. Clin Cancer Res, 2000, 6(11):4278～4286
1 易文君，唐中华，陈干农，等．乳腺癌VEGF，bFGF及其受体的表达和与微血管计数的关系[J]．中国现代医学杂志，2003，13(20)：39～13
2 Thompson M A, Witzig T E, Kumar S, et al. Plasma levels of tumour necrosis factor alpha and interleukin-6 predict progression-free survival following tha-lidomide therapy in patient s wit hpreviously unt reated multiple myeloma [J]. Br J Haematol, 2003, 123 (2):305～308
3 Zhang N, Ahsan M H, Zhu L, et al. NF-κ B and not the MAPK Signaling Pathway Regulates GADD45 β Expression during Acute Inflammation [J]. J Biolo Chem, 2005, 280(22): 21400～21408
4 Michael EF. Thalidomide [J]. The Lancet, 2004, 363:1802
5 Dredge K, Dalgleish A G, Marriot t J B. Thalidomide analogs as emerging anti-cancer drugs [J]. Anticancer Drugs, 2003, 14 (5):331～335
6 Verheul H M, Panigrahy D, Yuan J, et al. Combination oral an2 tiangiogenict herapy with tha-lidomide and sulindac inhibis tumour growth in rabbits [J]. Br J Cancer, 1999, 79 (1):114～118
7 张中林，刘志苏，孙权．反应停对人肝细胞癌生长侵袭的作用[J]．中华实验杂志，2005，22(8)：933～935
8 季艳霞，姜达，康振桥．沙立度胺对人乳腺癌细胞增殖及血管内皮生长因子表达的影响[J]．肿瘤学杂志，2005，11(1)：26～28
9 Stephens TD, Bunde CJ, Fillmore BJ. Mechanism of action in thalidomide teratogenesis [J]. Biochem Pharmacol, 2000, 59(12): 1489～1499
10 Yabu T, Tomimoto H, Taguchi Y, et al. Thalidomide-induced anti-angiogenic action is mediated by ceramide through depletion of VEGF receptors, and antagonized by sphingosine-1-phosphate [J]. Blood, 2005, 10:1182
11 Fujita J. Thalidomide and its analogues inhibite lipopoly saccharide-mediated induction of cyclooxygensase-2 [J]. Clin Cancer Res, 2001, 7:3349
12 Settles B, Stevenson A, Wilson K, et al. Down regulation of cell adhesion molecules LFA-1 and ICAM-1 after in vivo treatment with the anti-TNF-alpha agent thalidomide [J]. Cell Mol Biol (Noisy-le-grand), 2001, 47(7):1105～1114
13 Arrieta O, Guevara P, Tamarize J, et al. Antiproliferative effect of thlidomide alone and combined with carmusitine against C6 rat glioma [J]. Int J Exp Pathol, 2002, 83(2): 99～104
14 Marriott JB, Clarke IA, Czajka A, et al. A novel subclass of thalidomide analogue with anti-solide tumor activity in which caspase-dependent apoptosis is associated with altered expression of bcl-2 family proteins [J]. Cancer Res, 2003, 63 (2):593～599
15 Fine HA, Wen PY, Maher EA, et al. Phase II trail of thalidomide and carmustine for patiens with recurrent highgrade gliomas [J]. Clin Oncol, 2003, 21(12):2299～2304
16 Baumann F, Bjelijac M, Kollias SS, et al. Combined thalidomide and temozolmide treatment in pationts with glioblastomamultiforme [J]. Neurooncol, 2004, 67(1-2): 191～200
17 Motzer RJ, Berg W, Ginsberg M, et al. Phase II trial of thalidomide for patients with advanced renal cell carcinoma [J]. Clin Oncol, 2002, 20(1):302～306
18 Amato RJ. Renal cell carcinoma: review of novel single- agent therapeutics and combination regimens [J]. Annals of Oncology, 2005, 16:7～15
19 Amato RJ. Phase II study of thalidomide + interleukin-2 in patients with metastatic renal cell carcinoma [J]. Proc Am Soc Clin Oncol, 2003, 22:387
20 Kedar I, Mermershtain W, Ivgi H. Thalidomide reduces serum Creactive protein and interleukin-6 and induces response to IL-2 in afraction of metastatic renal cell cancer patiens who failed IL-2-based therapy [J]. Int J cancer, 2004, 110(2):260～265
21 Drake MJ. An open-label phase II study of low-dose thalidomide in androgen-independent prostate cancer [J]. Br J Cancer, 2003, 88(6):822
22 Dahut WL. Randomized phase II trial of docetaxel plus thalidomide in androgen-independent prostate cancer [J]. J Clin Oncol, 2004, 22(3):2532
23 Hwu WJ, Krown SE, Panageas KS, et al. Temozolomide plus thalidomide in patients with advanced melanoma: results of a dose-finding trial [J]. J Clin Oncol, 2002, 20 (11): 2610～2615
24 Danson S, Lorigan P, Arance A, et al. Randomized phase II study of temozolomide given every 8 hours or daily with either interferon alfa-2b or thalidomide in metastatic malignant melanoma [J]. J Clin Oncol, 2003, 21(13): 2551～2557
25 Soni S, Lee DS, Divito J Jr, et al. Treatment of pediatric ocular melanoma with high-dose interleukin-2 and thali- domide [J]. Pediatr Hematol Oncol, 2002, 24(6):488～491
26 Little RF, Wyvill KM, Pluda JM, et al. Activity of thalido-mide in AIDS-related Kappsi's sarcoma [J]. Clin Oncol, 2000, 18(13):2593～2602
27 Eisen T, Boshoff C, Mark I, et al. Continuous low dose thalidomide: a phase II study in advanced melanoma, renal cell, ovarian and breast cancer [J]. Br J Cancer, 2000, 82(1): 812～817
28 Govindarajan R. Irinotecan and thalidomide in metastatic colorectal cancer [J]. Oncolgy (Huntingt), 2000, 14(12～13): 29～32
29 Wang TE. Salvage therapy for hepatocellular carcinoma with thalidomide [J]. World J Gastroenterol, 2004, 10 (5):649
30 Zhu A X, Fuchs C S, Clark J W. A Phase II Study of Epirubicin and Thalidomide in Unresectable or Metastatic Hepatocellular Carcinoma [J]. Oncologist, 2005, 10:392～398
31 Zangari M. Thrombogenic activity of doxorubicin in myeloma patients receiving thalidomide: Implications for therapy [J]. Blood, 2002, 100:1168
32 Zangari M. Deep vein thrombosis in patients with multiple myeloma treated with thalidomide and chemotherapy effects of prophylactic and therapeutic anticoagulation [J]. Br J Haematol, 2004, 126(5):715
33 Zhu A X. Response to"Acute Tumor Lysis Syndrome After Thalidomide Therapy in Advanced Hepatocellular Carcinoma" [J]. Oncologist, 2006, 11:89