摘要
目的构建针对肿瘤相关基因AKT1、COX-2和PI3Kp85基因的shRNA重组腺病毒质粒表达载体,检测其在胃腺癌细胞株SGC-7901中对AKT1、COX-2和PI3Kp85基因表达的抑制作用,以及敲低靶基因的表达后,观察MMP-2、MMP-9、TIMP-2和VEGF的表达变化。在体外实验中检测肿瘤细胞侵袭和迁移活性的变化,并进一步探讨COX-2和PI3K/AKT信号通路在胃腺癌侵袭迁移过程中的作用机制。
方法本课题分两部分进行:
第一部分设计可形成小干扰RNA的AKT1、COX-2和PI3Kp85 shRNA对应模板DNA序列,克隆至线性化质粒PEGFP6-1、PGENESIL-2、PEGFP6-4上,构建一个编码AKT1、COX-2和PI3Kp85三条shRNA的质粒表达载体。将此质粒亚克隆至腺病毒表达载体上,经酶切、测序鉴定正确后包装扩增并鉴定其病毒滴度,然后转染至SGC7901细胞中检测其转染率。
第二部分在体外应用RNA干涉技术,以构建的重组腺病毒介导靶向AKT1、COX-2和PI3Kp85的shRNA转染人胃腺癌细胞系SGC-7901后,应用Realtime PCR和Western blot法检测目的基因的敲低情况,并用Western blot法检测RNAi敲低靶基因的表达后,COX-2及EGFR通路下游侵袭相关因子MMP-2、MMP-9、TIMP-2和VEGF的蛋白表达变化。应用ELISA检测转染前后分泌到细胞外的MM9-2和MMP-9的浓度变化。应用划痕实验、Transwell实验、2-D和3-D的Matrigel基质生长实验评价肿瘤细胞转染前后侵袭迁移能力的变化。
结果
1.通过应用定向克隆、同源重组等技术,获得一种同时包含靶向COX-2、PI3Kp85和AKT1三条RNA干扰序列的真核表达质粒,经酶切分析,测序鉴定表明构建成功后,扩增并亚克隆至重组腺病毒载体上,获得重组腺病毒质粒表达载体rAd5-C-A-P。由于重组腺病毒质粒表达载体上带有荧光蛋白表达框,所以通过转染SGC-7901后观察荧光蛋白表达情况,计算转染率>90%。
2. Realtime PCR和Western blot结果提示,转染AKT1、COX-2和PI3Kp85 shRNA后可明显敲低靶基因mRNA和蛋白的表达。在靶基因被下调后通过Western blot检测发现COX-2及PI3K/AKT信号通路下游侵袭相关因子MMP-2、MMP-9和VEGF的表达水平均明显降低,而TIMP-2表达上调。应用ELASA检测发现rAd5-C-A-P转染组细胞培养液中MMP-2和MMP-9明显减低,划痕实验显示与对照组和无义序列组相比,rAd5-C-A-P转染组细胞迁移运动能力明显减弱,Transwell体外侵袭实验结果显示对照组和无义序列组穿过细胞数分别为(105±4)、(102.5±6.4),而rAd5-C-A-P转染组(26.4±4.6) (p<0.001).2-D的Matrigel基质生长实验显示对照组和无义序列组细胞呈正常形态贴壁生长并融合成片,而rAd5-C-A-P转染组细胞贴壁能力与迁移能力均明显减低,3-D的Matrigel基质生长实验显示与对照组和无义序列组比较,rAd5-C-A-P转染组细胞形成的细胞团块较小(p<0.001)。
结论
1.成功构建靶向AKT1、COX-2和PI3Kp85的重组腺病毒质粒表达载体,其能在体外高效转染肿瘤细胞。
2.靶向AKT1、COX-2和PI3Kp85的RNA干扰技术可以序列特异性地抑制SGC7901细胞的AKT1、COX-2和:PI3Kp85表达,在体外实验中对胃腺癌的侵袭迁移起到了明显的抑制作用。因此靶向AKT1、COX-2和PI3Kp85的联合基因治疗有望成为胃癌基因治疗的新措施。
AIM The aim of this study was to construct a recombinant adenovirus vector that expresses small hairpin RNA against the COX-2, AKT1 and PI3Kp85 gene and to detect the suppression effect on the expression of COX-2, AKT1 and PI3Kp85 in gastric adenocarcinoma cell SGC-7901. And after COX-2, AKT1 and PI3Kp85 were down-regulated, we examined the effects of COX-2, AKT1 and PI3Kp85 shRNA on the invasion and metastasis of gastric adenocarcinoma, and the effects of the down-regulation of COX-2, AKT1 and PI3Kp85 on expression of MMP-2, MMP-9, TIMP-2 and VEGF. The mechanism of COX-2 and PI3K/AKT signal pathway in the invasion and metastasis of gastric adenocarcinoma is further investigated.
METHODS The study was divided into 2 parts.
1. AKT1+COX-2+PI3Kp85shRNA template DNA sequence, capable of forming a small hairpin structure was designed. After renaturation, cloned into the linearisation plasmid PEGFP6-1, PGENESIL-2, PEGFP6-4, then construct a plasmid expression vector, which coding three strip of shRNA. Subcloned the AKT1+COX-2+PI3Kp85 shRNA expression frame into adenovirus expression vector. After verification, enclose、amplify and titered, then transfected the SGC7901 cell to detect the transfection efficiency.
2. The recombinant adenovirus vector plasmid expression vector which contained short hairpin RNA (shRNA) expression construct targeting COX-2, AKT1 and PI3Kp85 was transfected into human gastric adenocarcinoma SGC7901 cells. Realtime PCR and Western blot analysis were used to detecte COX-2, AKT1 and PI3Kp85 expression. And the expression of MMP-2, MMP-9, TIMP-2 and VEGF were also detected by Western blot when COX-2, AKT1 and PI3Kp85 were downregulated. ELESA was used to detecte the ectocytic density change of MMP-2 and MMP-9.The invasion and metastasis ability of the tumor cells were measured by Scarification Transwell、2-dementinal and 3-dementional matrigel matrix.
RESULTS
1. By using directional cloning, homologous recombination techniques, to obtain a plasmid which includes three shRNA sequence targeting COX-2, PI3Kp85 and AKT1. Restriction digest patterns and squencing analysis showed that rAd5-C-A-P adenovirus expression vector was constructed successfully. And the expression vector contained fluorescin expression frame, so we detected the transfection efficiency by observing the expression of fluorescin after transfection. And the transfection efficiency was over 90%.
2. Recombinant adenovirus vector rAd5-C-A-P mediated shRNA targeting COX-2, AKT1 and PI3Kp85 dramatically down-regulated their expression in SGC7901 cells. And MMP-2, MMP-9 and VEGF were also downregulated, while TIMP-2 was upregulated. The ectocytic density of MMP-2, and MMP-9 decreased. Scarification indicates that compared with control group and nonsense sequence group the ability of invision was decreased obviously. Transwell showed that the number of cells invading through the matrigel was control group (105±4), nonsense sequence group (102.5±6.4). rAd5-C-A-P transfection group (26.4±4.6) (p<0.001). Cell growth on matrigel matrix showed normal cell appearances and mix together in control group and nonsense sequence group cells, while the cells of rAd5-C-A-P transfection group were detached from the matrix or grew in a scattered clustering patterns, signifying poor cell metastasis activities in 2-D matrigel. The rAd5-C-A-P transfection group cells formed only small aggregates as compared with the control group and nonsense sequence group in 3-D matrigel matrix growth(p<0.001).
CONCLUSION
1. Recombinant adenovirus vector expression vector targeting COX-2, AKT1 and PI3KP85 was constructed successfully, and it may transfect the tumor cell with high efficiency.
2. ShRNA targeting COX-2, AKT1 and PI3KP85 downregulates significantly their expression in a sequence-specific manner, exerted invasion and metastasis inhibition effect on SGC7901 cells in vitro. Consequently, combined gene therapy by targeting COX-2, AKT1 and PI3KP85 would be a new strategy in targeting gene therapy of gastric adenocarcinoma.
引文
[1]Kim JW, Hwang I, Kim MJ, et al. Clinic pathological characteristics and predictive markers of early gastric cancer with recurrence[J]. J Korean Med Sci. 2009,24(6):1158-1164.
[2]Iwamoto J, Mizokami Y, Takahashi K, et al. The effects of cyclooxygenase2-prostaglandinE2 pathway on Helicobacter pylori-induced urokinase-type plasminogen activator system in the gastric cancer cells[J]. Helicobacter.2008,13(3):174-182.
[3]Sitarz R, Leguit RJ, de Leng WW, et al. Cyclooxygenase-2 mediated regulation of E-cadherin occurs in conventional but not early-onset gastric cancer cell lines[J]. Cell Oncol.2009,31(6):475-485.
[4]Kim N, Kim CH, Ahn DW, et al. Anti-gastric cancer effects of celecoxib, a selective COX-2 inhibitor, through inhibition of Akt signaling[J]. J Gastroenterol Hepatol.2009,24(3):480-487
[5]Huang MT, Chen ZX, Wei B, et al. The inhibitive effects of celecoxib combined with octreotide on the metastasis of human gastric cancer in vivo[J]. Zhonghua Yi Xue Za Zhi.2006,86(46):3255-3259.
[6]Mrena J, Wiksten JP, Nordling S, et al. MMP-2 but not MMP-9 associated with COX-2 and survival in gastric cancer. J Clin Pathol.2006,59(6):618-623.
[7]Dicken BJ, Graham K, Hamilton SM, et al. Lymphovascular invasion is associated with poor survival in gastric cancer:an application of gene-expression and tissue array techniques[J]. Ann Surg.2006,243(1):64-73.
[8]Sun WH, Sun YL, Fang RN, et al. Expression of cyclooxygenase-2 and matrix metalloproteinase-9 in gastric carcinoma and its correlation with angiogenesis[J]. Jpn J Clin Oncol.2005,35(12):707-713.
[9]Xu K, Shu HK. EGFR activation results in enhanced cyclooxygenase-2 expression through p38 mitogen-activated protein kinase-dependent activation of the Sp1/Sp3 transcription factors in human gliomas[J]. Cancer Res.2007, 67(13):6121-6129.
[10]Wu R, Coniglio SJ, Chan A, Symons MH, Steinberg BM. Up-regulation of Racl by epidermal growth factor mediates COX-2 expression in recurrent respiratory papillomas[J]. Mol Med.2007,13(3-4):143-150.
[11]Yang L, Amann JM, Kikuchi T, Porta R, Guix M, Gonzalez A, Park KH, Billheimer D, Arteaga CL, Tai HH, DuBois R, Carbone DP, Johnson DH. Inhibition of epidermal growth factor receptor signaling elevates 15-hydroxyprostaglandin dehydrogenase in non-small-cell lung cancer[J]. Cancer Res.2007,67(12):5587-5593.
[12]Subbaramaiah K, Dannenberg AJ. Cyclooxygenase-2 transcription is regulated by human papillomavirus 16 E6 and E7 oncoproteins:evidence of a corepressor/coactivator exchange[J]. Cancer Res.2007,67(8):3976-3985.
[13]Lev-Ari S, Starr A, Vexler A, Karaush V, Loew V, Greif J, Fenig E, Aderka D, Ben-Yosef R. Inhibition of pancreatic and lung adenocarcinoma cell survival by curcumin is associated with increased apoptosis, down-regulation of COX-2 and EGFR and inhibition of Erkl/2 activity[J]. Anticancer Res.2006, 26(6B):4423-4430.
[14]Wu R, Abramson AL, Shikowitz MJ, et al. Epidermal growth factor-induced cyclooxygenase-2 expression is mediated through phosphatidylinositol-3 kinase, not mitogen-activated protein/extracellular signal-regulated kinase kinase, in recurrent respiratory papillomas[J]. Clin Cancer Res.2005,11(17):6155-6161.
[15]Han C, Michalopoulos GK, Wu T. Prostaglandin E2 receptor EP1 transactivates EGFR/MET receptor tyrosine kinases and enhances invasiveness in human hepatocellular carcinoma cells[J]. J Cell Physiol.2006, 207(1):261-270.
[16]Pai R, Soreghan B, Szabo IL, et al. Prostaglandin E2 transactivates EGF receptor: a novel mechanism for promoting colon cancer growth and gastrointestinal hypertrophy[J]. Nat Med.2002,8:289-293.
[17]Bernstein E, Caudy AA, Hammond SM, et al. Role for a bidentate ribonuclease in the initiation step of RNA interference[J]. Nature.2001; 409:363-366.
[18]Hattmrth J, Elbasbir SM, Weber K et al. Identification ofessential genes in cultured mammalian cells using small interfering RNAs. Journal of Cell Science, 2001,114(Pt 24):4557-4565
[19]Gupta RA, Dubois RN. Colorectal cancer prevention and treatment by inhibition of cyclooxygenase-2[J]. Nat Rev Cancer,2001,1:11-21.
[20]Elbashir SM, Harborth J, Weber K et al. Analysis of gene function in somatic mammalian cells using small interfering RNAs[J]. Methods,2002, 26(2):199-213.
[21]Sobol RE, Scanlon KJ. Cancer gene therapy clinical trials[J]. Cancer Gene Ther JT-Cancer gene therapy,1995,2(1):5-6.
[22]Javle MM, Yu J, Khoury T, et al. Akt expression may predict favorable prognosis in cholangiocarcinoma[J]. J Gastroenterol Hepatol.2006, 21(11):1744-1751.
[23]Xu K, Shu HK. EGFR activation results in enhanced cyclooxygenase-2 expression through p38 mitogen-activated protein kinase-dependent activation of the Spl/Sp3 transcription factors in human gliomas[J]. Cancer Res.2007, 67(13):6121-6129.
[24]Shao J, Lee SB, Guo H, et al. Prostaglandin E2 stimulates the growth of colon cancer cells via induction of amphiregulin[J]. Cancer Res.2003,63:5218-5223.
[25]Elbashir SM, Martinez J, Patkaniowska A, et al. Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate[J]. EMBO J JT-The EMBO journal,2001,20(23):6877-6888.
[26]Shen C, Buck AK, Liu X, et al.Gene silencing by adenovirus-delivered siRNA[J].FEBS Lett.2003,539(1-3):111-114
[27]Dannenberg AJ, Altorki NK, Boyle JO, et al. Cyclooxygenase2:a pharmacological target for theprevention of cancer[J]. Lancet Oncol.2001,2: 544-551.
[28]Gupta RA, Dubois RN. Colorectal cancer prevention and treatment by inhibition of cyclooxygenase-2 [J]. Nat Rev Cancer.2001,1:11-21.
[29]Cully M, You H, Levine AJ and Mak TW. Beyond PTEN mutations:the PI3K pathway as an integrator of multiple inputs during tumorigenesis[J]. Nat Rev Cancer.2006,6:184-192.
[30]Vivanco I and Sawyers CL.The phosphatidylinositol 3-kinase AKT pathway in human cancer[J]. Nat Rev Cancer.2002,2:489-501.
[31]Fingar DC and Blenis J. Target of rapamycin (TOR):an integrator of nutrient and growth factor signals and coordinator of cell growth and cell cycle progression[J]. Oncogene.2004,23:3151-3171.
[32]Duan C, Bauchat JR and Hsieh T. Phosphatidylinositol 3-kinase is required for insulin-like growth factor-I-induced vascular smooth muscle cell proliferation and migration[J]. Circ Res.2000,86:15-23.
[33]Cartwright JE, Tse WK and Whitley GS. Hepatocyte growth factor induced human trophoblast motility involves phosphatidylinositol-3-kinase, mitogen-activated protein kinase, and inducible nitric oxide synthase[J]. Exp Cell Res.2002,279:219-226.
[34]Kim MS, You MK, Rhuy DY, et al. Loquat (Eriobotrya japonica) extracts suppress the adhesion, migration and invasion of human breast cancer cell line[J]. Nutr Res Pract.2009,3(4):259-264.
[35]Wang ZD, Huang C, Li ZF, et al. Chrysanthemum indicum ethanolic extract inhibits invasion of hepatocellular carcinoma via regulation of MMP/TIMP balance as therapeutic target[J]. Oncol Rep.2010,23 (2):413-421.
[36]Roomi MW, Monterrey JC, Kalinovsky T, et al. Modulation of MMP-2 and MMP-9 by cytokines, mitogens and inhibitors in lung cancer and malignant mesothelioma cell lines[J]. Oncol Rep.2009,22(6):1283-1291.
[37]Mott JD, Werb Z. Regulation of matrix biology by matrix metalloproteinases[J]. Curr. Opin. Cell Biol.2004,16(5):558-564.
[38]Nagase H, Visse R, Murphy G. Structure and function of matrix metalloproteinases and TIMPs[J]. Cardiovasc Res.2006,69(3):562-573.
[39]Cawston TE, Wilson AJ.Understanding the role of tissue degrading enzymes and their inhibitors in development and disease[J]. Best Pract Res Clin Rheumatol. 2006,20(5):983-1002.
[40]Wang ZD, Huang C, Li ZF, et al. Chrysanthemum indicum ethanolic extract inhibits invasion of hepatocellular carcinoma via regulation of MMP/TIMP balance as therapeutic target[J]. Oncol Rep.2010,23 (2):413-421.
[41]Zhang X, Wang Y, Yamamoto G, et al.11.Expression of matrix metalloproteinases MMP-2, MMP-9 and their tissue inhibitors TIMP-1 and TIMP-2 in the epithelium and stroma of salivary gland pleomorphic adenomas[J]. Histopathology.2009,55(3):250-260.
[42]Yan S, Zhou C, Lou X, et al. PTTG overexpression promotes lymph node metastasis in human esophageal squamous cell carcinoma[J]. Cancer Res.2009, 69(8):3283-3290.
[43]Baker EA, Stephenson TJ, Reed MWR, et al. Expression of proteinases and inhibitors in human breast cancer progression and survival[J]. Molecular Pathology,2002,55 (5):300-304.
[44]Wu LM, Xie HY, Zhou L, et al. A single nucleotide polymorphism in the vascular endothelial growth factor gene is associated with recurrence of hepatocellular carcinoma after transplantation[J]. Arch Med Res.2009, 40(7):565-570.
[45]Raskopf E, Vogt A, Standop J, et al. Inhibition of neuropilin-1 by RNA-interference and its angiostatic potential in the treatment of hepatocellular carcinoma[J]. Z Gastroenterol.2010,48(1):21-27
[46]Tall en G, Farhangi S, Tamannai M, et al. The inhibitor of growth 1 (ING1) proteins suppress angiogenesis and differentially regulate angiopoietin expression in glioblastoma cells[J]. Oncol Res.2009,18(2-3):95-105.
[47]Lee SJ, Kim JG, Sohn SK, et al. No Association of Vascular Endothelial Growth Factor-A (VEGF-A) and VEGF-C Expression with Survival in Patients with Gastric Cancer[J]. Cancer Res Treat.2009,41(4):218-223.
[48]Kosem M, Tuncer I, Kotan C, etal. Significance of VEGF and microvascular density in gastric carcinoma [J]. Hepatogastroenterology.2009, 56(93):1236-1240.
[49]Janusz Rak, Joanne L Yu. Oncogenes and tumor angiogenes is:The question of vascular supply and vascular demand[J]. Seminarsin Cancer Biology,2004,14(2): 93-104.
[50]Nyberg P, Xie L, Kalluri R. Endogenous inhibitors of angiogenesis [J]. Cancer Res,2005,65(10):3967-3979.
[51]Batmunkh E, Shimada M, Morine Y, et al. Expression of hypoxia-inducible factor-1 alpha (HIF-1alpha) in patients with the gallbladder carcinoma[J]. Int J Clin Oncol.2010,16(4)132-141.
[52]Luo H, Rankin GO, Liu L, et al. Kaempferol inhibits angiogenesis and VEGF expression through both HIF dependent and independent pathways in human ovarian cancer cells [J]. Nutr Cancer.2009,61(4):554-563.
[53]Molitoris KH, Kazi AA, Koos RD. et al. Inhibition of oxygen-induced hypoxia-inducible factor-1 alpha degradation unmasks estradiol induction of vascular endothelial growth factor expression in ECC-1 cancer cells in vitro[J]. Endocrinology.2009,150(12):5405-5414.
[54]Chien MH, Ku CC, Johansson G, et al. Vascular endothelial growth factor-C (VEGF-C) promotes angiogenesis by induction of COX-2 in leukemic cells via the VEGF-R3/JNK/AP-1 pathway[J]. Carcinogenesis.2009,30(12):2005-2013.
[55]Liu H, Yang Y, Xiao J, et al. Inhibition of cyclooxygenase-2 suppresses lymph node metastasis via VEGF-C[J]. Anat Rec (Hoboken).2009,292(10):1577-1583.
[56]Spinella F, Rosano L, Di CV et al. Endothelin-1-induced prostaglandinE2-EP2, EP4 signaling regulates vascular endothelial growth factor production and ovarian carcinoma cell invasion[J]. J BiolChem,2004,279:46700-46705.
[57]Pan J, Kong L, Lin S, etal.The clinical significance of coexpression of cyclooxygenases-2, vascular endothelial growth factors, and epidermal growth factor receptor in nasopharyngeal carcinoma[J]. Laryngoscope.2008, 118(11):1970-1975.
[58]Scandlyn MJ, Stuart EC, Somers-Edgar TJ, etal. A new role for tamoxifen in oestrogen receptor-negative breast cancer when it is combined with epigallocatechin gallate. Br J Cancer.2008,99(7):1056-1063.
[59]Saad AG, Yeap BY, Thunnissen FB, etal.Immunohistochemical markers associated with brain metastases in patients with nonsmall cell lung carcinoma[J]. Cancer.2008,113(8):2129-2138.
[60]Meteoglu I, Erdogdu IH, Meydan N, etal. NF-KappaB expression correlates with apoptosis and angiogenesis in clear cell renal cell carcinoma tissues[J]. J Exp Clin Cancer Res.2008,114(19)27:53.
[61]Chang BD, Cao LS, Zhou HL. Expression of COX-2 and VEGF and their correlation with angiogenesis in human clear cell renal cell carcinoma[J]. Zhonghua Zhong Liu Za Zhi.2009,31(9):687-690.
[62]Chien MH, Ku CC, Johansson G, et al. Vascular endothelial growth factor-C (VEGF-C) promotes angiogenesis by induction of COX-2 in leukemic cells via the VEGF-R3/JNK/AP-1 pathway[J]. Carcinogenesis.2009,30(12):2005-2013.
[63]Wei J, Yan W, Li X, et al. Thromboxane receptor alpha mediates tumor growth and angiogenesis via induction of vascular endothelial growth factor expression in human lung cancer cells[J]. Lung Cancer.2009,23(6):112-121.
[1]Baltazar F, Filho AL, Pinheiro C, et al. Cyclooxygenase-2 and epidermal growth factor receptor expressions in different histological subtypes of cervical carcinomas[J]. Int J Gyneccol.2007,26(3):235-41.
[2]Lee TS, Jeon YT, Kim JW, et al. Increased cyclooxygenase-2 expression associated with inflammatory cellular infiltration in elderly patients with vulvar cancer[J]. Ann N Y Acad Sci.2007,1095(6):143-153.
[3]Fang FM, Li CF, Chien CY, et al. Immunohistochemical expression of epidermal growth factor receptor and cyclooxygenase-2 in pediatric nasopharyngeal carcinomas:no significant correlations with clinicopathological variables and treatment outcomes[J]. Int J Pediatr Otorhinolaryngol.2007,71(3):447-455.
[4]Foster J, Black J, LeVea C, et al. COX-2 expression in hepatocellular carcinoma is an initiation event; while EGF receptor expression with downstream pathway activation is a prognostic predictor of survival[J]. Ann Surg Oncol.2007,14(2):752-758.
[5]Javle MM, Yu J, Khoury T, et al. Akt expression may predict favorable prognosis in cholangiocarcinoma[J]. J Gastroenterol Hepatol.2006, 21(11):1744-1751.
[6]Hakozaki M, Hojo H, Sato M, et al. Establishment and characterization of a new cell line, FPS-1, derived from human undifferentiated pleomorphic sarcoma, overexpressing epidermal growth factor receptor and cyclooxygenase-2[J]. Anticancer Res.2006,26(5A):3393-3401.
[7]Palmieri G, Marino M, Salvatore M, et al. Cetuximab is an active treatment of 'metastatic and chemorefractory thymoma[J]. Front Biosci.2007,12:757-761.
[8]Rieker RJ, Joos S, Mechtersheimer G, et al. COX-2 upregulation in thymomas and thymic carcinomas [J]. Int J Cancer.2006,119(9):2063-2070.
[9]Li Y, Wo JM, Ray MB, et al. Cyclooxygenase-2 and epithelial growth factor receptor up-regulation during progression of Barrett's esophagus to adenocarcinoma[J]. World J Gastroenterol.2006,12(6):928-934.
[10]Hakozaki M, Hojo H, Sato M, et al. Establishment and characterization of a new cell line, FRTK-1, derived from human malignant rhabdoid tumor of the kidney, with overexpression of epidermal growth factor receptor and cyclooxygenase-2[J]. Oncol Rep.2006,16(2):265-271.
[11]Xu K, Shu HK. EGFR activation results in enhanced cyclooxygenase-2 expression through p38 mitogen-activated protein kinase-dependent activation of the Spl/Sp3 transcription factors in human gliomas[J]. Cancer Res.2007, 67(13):6121-6129.
[12]Wu R, Coniglio SJ, Chan A, et al. Up-regulation of Racl by epidermal growth factor mediates COX-2 expression in recurrent respiratory papillomas[J]. Mol Med.2007,13(3-4):143-150.
[13]Wu R, Abramson AL, Shikowitz MJ, et al. Epidermal growth factor-induced cyclooxygenase-2 expression is mediated through phosphatidylinositol-3 kinase, not mitogen-activated protein/extracellular signal-regulated kinase kinase, in recurrent respiratory papillomas[J]. Clin Cancer Res.2005,11(17):6155-6161.
[14]Yang L, Amann JM, Kikuchi T, et al. Inhibition of epidermal growth factor receptor signaling elevates 15-hydroxyprostaglandin dehydrogenase in non-small-cell lung cancer[J]. Cancer Res.2007,67(12):5587-5593.
[15]Lev-Ari S, Starr A, Vexler A, et al. Inhibition of pancreatic and lung adenocarcinoma cell survival by curcumin is associated with increased apoptosis, down-regulation of COX-2 and EGFR and inhibition of Erkl/2 activity[J]. Anticancer Res.2006,26(6B):4423-4430.
[16]Halrf E, Sun Y, Sinicrope FA. Anti-EGFR and ErbB-2 antibodies attenuate cyclooxygenase-2 expression and cooperatively inhibit survival of human colon cancer cells[J]. Cancer Lett.2007,251(2):237-246.
[17]Eblin KE, Bredfeldt TG, Buffington S, et al. Mitogenic signal transduction caused by monomethylarsonous acid in human bladder cells:role in arsenic-induced carcinogenesis[J]. Toxicol Sci.2007,95(2):321-330.
[18]Han C, Michalopoulos GK, Wu T. Prostaglandin E2 receptor EP1 transactivates EGFR/MET receptor tyrosine kinases and enhances invasiveness in human hepatocellular carcinoma cells[J]. J Cell Physiol.2006, 207(1):261-270.
[19]Pai R, Soreghan B, Szabo IL, et al. Prostaglandin E2 transactivates EGF receptor:a novel mechanism for promoting colon cancer growth and gastrointestinal hypertrophy[J]. Nat Med2002,8:289-93.
[20]Shao J, Lee SB, Guo H, et al. Prostaglandin E2 stimulates the growth of colon cancer cells via induction of amphiregulin[J]. Cancer Res 2003, 63:5218-23.
[21]Banu N, Buda A, Chell S, et al. Inhibition of COX-2 with NS-398 decreases colon cancer cell motility through blocking epidermal growth factor receptor transactivation:possibilities for combination therapy[J]. Cell Prolif. 2007,40(5):768-779.
[22]Dougherty U, Sehdev A, Cerda S et al. Epidermal growth factor receptor controls flat dysplastic aberrant crypt foci development and colon cancer progression in the rat azoxymethane model[J]. Clin Cancer Res.2008, 14(8):2253-62.
[23]Buchanan FG, Holla V, Katkuri S, et al. Targeting cyclooxygenase-2 and the epidermal growth factor receptor for the prevention and treatment of intestinal cancer[J]. Cancer Res.2007,67 (19):9380-9388.
[24]Lanza-Jacoby S, Burd R, Rosato FE Jr, et al. Effect of simultaneous inhibition of epidermal growth factor receptor and cyclooxygenase-2 in HER-2/neu-positive breast cancer[J]. Clin Cancer Res.2006,12 (20 Pt 1):6161-6169.
[25]Ali S, El-Rayes BF, Sarkar FH, et al. Simultaneous targeting of the epidermal growth factor receptor and cyclooxygenase-2 pathways for pancreatic cancer therapy[J]. Mol Cancer Ther.2005,4(12):1943-1951.
[26]Zhang X, Chen ZG, Choe MS, et al. Tumor growth inhibition by simultaneously blocking epidermal growth factor receptor and cyclooxygenase-2 in a xenograft model[J]. Clin Cancer Res.2005, 11(17):6261-6269.
[27]Melisi D, Caputo R, Damiano V, et al. Zoledronic acid cooperates with a cyclooxygenase-2 inhibitor and gefitinib in inhibiting breast and prostate cancer[J]. Endocr Relat Cancer.2005,12(4):1051-1058.