phTERT-tumstatin载体构建及其抗血管形成
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摘要
肝癌细胞的无限增殖和新生血管的不断形成是肝癌难以有效控制的重要原因。肝癌细胞端粒酶的激活是其无限增殖的基础,研究证实肝癌细胞中端粒酶活性高表达源于其hTERT(端粒酶逆转录酶)基因启动子的完全开启。血管生成是肿瘤存活和转移的关键过程,靶向抑制肿瘤新生血管的生成可间接抑制肿瘤的生长和转移。HCC是高度血管化的肿瘤,因此利用肝癌细胞特异的hTERT启动子驱动抗血管形成基因以达到靶向抑制肝癌新生血管的形成将是一个有前景的治疗策略。
目的:
构建由hTERT启动子驱动的肿瘤抑素基因载体,即phTERT-tumstatin质粒,脂质体方法转染入肝癌细胞,观察由hTERT启动子驱动的肿瘤抑素基因在肝癌细胞内特异地表达、分泌及其体外抗血管形成效应。
方法:
1.体外培养人肝癌细胞HepG2、人正常肝细胞L-02、人血管内皮细胞HUVEC。2.构建phTERT-tumstatin质粒以及对照组质粒; 3.脂质体转染HepG2、L-02细胞; 4.荧光显微镜检测EGFP的表达, Western blotting检测目的基因Tumstatin在HepG2细胞的表达、分泌;5. MTS法检测稳定转染后HepG2细胞的增殖活性; 6. MTS法检测含或不含目的蛋白的条件培养基对HUVEC细胞增殖活力的影响。7.通过计数内皮细胞在基质胶上形成的管样分支数观察目的蛋白对HUVEC细胞管道结构形成的影响。
结果:
1.成功构建了质粒phTERT-tumstatin、pCMV-tumstatin(阳性对照)、phTERT-EGFP(阴性对照);2.pCMV-tumstatin质粒在HepG2 和 L-02细胞中均有荧光表达(图2-1、2-2),phTERT-tumstatin仅在HepG2细胞中有荧光表达,L-02细胞中几乎无表达,说明hTERT启动子仅在肿瘤细胞中才有驱动活性。3.Tumstatin基因在肝癌细胞HepG2中特异地表达、分泌,在正常肝细胞L-02中无表达;4.含目的蛋白Tumstatin的条件培养基CM-T抑制了HUVEC细胞的增殖,抑制率达(56.49±0.33)%;5.HUVEC细胞在添加了条件培养基CM-T的基质胶上形成的管样分支数为(3.33±1.53)%;CM-T与不含目的蛋白的条件培养基CM-N、CM-NT相比显著抑制了HUVEC细胞血管结构的形成【(3.33±1.53)% vs (24.44±3.11)%、(23.94±2.92)%,P<0.01)】。
结论:
phTERT-tumstatin质粒构建成功,选择性在肝癌细胞内表达和分泌的目的蛋白能明显抑制血管内皮细胞的增殖和血管结构的形成,对正常肝细胞几乎无影响,具有靶向抑制血管形成的效应。
Abstract
Unlimited proliferation of hepatoma cells and the continuous formation of new blood vessels are important reasons which are difficult to effectively control the Hepatic carcinoma. Hepatoma cell telomerase activation is the basis of their unlimited proliferation. Study confirmed that liver cancer cells with high expression of telomerase activity from the hTERT (human telomerase reverse transcriptase) gene promoter in the fully open. Angiogenesis is the key processes of tumor survival and metastasis. Targeted inhibition of tumor angiogenesis may indirectly inhibit tumor growth and metastasis. HCC is a highly vascularized tumor,therefore, using its specific hTERT promoter-driven anti-angiogenic genes to inhibit liver angiogenesis is a promising therapeutic strategy.
Objective
Construct the plasmid of phTERT-tumstatin gene vector driven by the hTERT promoter , To observe the specific expression and secretion of tumstatin transduced into HepG2 ,driven by the hTERT promoter and its anti-angiogenic effect in vitro.
Methods
1.Cells HepG2、L-02 and HUVEC were cultured in vitro cell culture. 2.Constructed phTERT-tumstatin plasmid and its control group. 3.We transfected HepG2 and L-02 with the plasmid of phTERT-tumstatin. 4.The expression of EGFP was examined with fluorescence microscope observations;The secretion of target protein tumstatin from HepG2 cells was detected by Western blotting. 5.MTS assay was used to measure cell proliferation of HepG2 cells after stably transfected by plasmids. 6.The effect of conditioned mediun (containing target protein or not) on the proliferation of HUVEC cells was detected by MTS assay. 7.The effect of target protein Tumstatin on the formation of HUVEC celluar tube structure can be observed through counting the number of tube branches.
Results
1.We successfully constructed phTERT-tumstatin, pCMV-tumstatin (positive control) and phTERT-EGFP(negative control) plasmids. 2.The specific expression and secretion of tumstatin was only observed in HepG2, but not in normal liver L-02 cells. 3.The target protein contained in CM-T inhibited the proliferation of HUVEC cells, inhibition rate reached (56.49±0.33)%. 4.The number of tube branches on Matrigel-coated plates supplemented with conditioned medium CM-T was (3.33±1.53)%. Compared with conditioned medium CM-N and CM-NT, supplement of CM-T significantly inhibited the cellular tube structure formation of HUVEC cells on Matrigel-coated plates【(3.33±1.53)% vs (24.44±3.11)%、(23.94±2.92)%,P<0.01)】。
Conclusion
The plasmid of hTERT-tumstatin was successfully constructed. The target protein selective expression and secretion in liver cancer cells can inhibit the proliferation of endothelial cells and vascular structure formation and almost have no influence on normal hepatic cells. The hTERT-tumstatin has a potential effect of targeted inhibition of angiogenesis .
目的:
构建由hTERT启动子驱动的肿瘤抑素基因载体,即phTERT-tumstatin质粒,脂质体方法转染入肝癌细胞,观察由hTERT启动子驱动的肿瘤抑素基因在肝癌细胞内特异地表达、分泌及其体外抗血管形成效应。
方法:
1.体外培养人肝癌细胞HepG2、人正常肝细胞L-02、人血管内皮细胞HUVEC。2.构建phTERT-tumstatin质粒以及对照组质粒; 3.脂质体转染HepG2、L-02细胞; 4.荧光显微镜检测EGFP的表达, Western blotting检测目的基因Tumstatin在HepG2细胞的表达、分泌;5. MTS法检测稳定转染后HepG2细胞的增殖活性; 6. MTS法检测含或不含目的蛋白的条件培养基对HUVEC细胞增殖活力的影响。7.通过计数内皮细胞在基质胶上形成的管样分支数观察目的蛋白对HUVEC细胞管道结构形成的影响。
结果:
1.成功构建了质粒phTERT-tumstatin、pCMV-tumstatin(阳性对照)、phTERT-EGFP(阴性对照);2.pCMV-tumstatin质粒在HepG2 和 L-02细胞中均有荧光表达(图2-1、2-2),phTERT-tumstatin仅在HepG2细胞中有荧光表达,L-02细胞中几乎无表达,说明hTERT启动子仅在肿瘤细胞中才有驱动活性。3.Tumstatin基因在肝癌细胞HepG2中特异地表达、分泌,在正常肝细胞L-02中无表达;4.含目的蛋白Tumstatin的条件培养基CM-T抑制了HUVEC细胞的增殖,抑制率达(56.49±0.33)%;5.HUVEC细胞在添加了条件培养基CM-T的基质胶上形成的管样分支数为(3.33±1.53)%;CM-T与不含目的蛋白的条件培养基CM-N、CM-NT相比显著抑制了HUVEC细胞血管结构的形成【(3.33±1.53)% vs (24.44±3.11)%、(23.94±2.92)%,P<0.01)】。
结论:
phTERT-tumstatin质粒构建成功,选择性在肝癌细胞内表达和分泌的目的蛋白能明显抑制血管内皮细胞的增殖和血管结构的形成,对正常肝细胞几乎无影响,具有靶向抑制血管形成的效应。
Abstract
Unlimited proliferation of hepatoma cells and the continuous formation of new blood vessels are important reasons which are difficult to effectively control the Hepatic carcinoma. Hepatoma cell telomerase activation is the basis of their unlimited proliferation. Study confirmed that liver cancer cells with high expression of telomerase activity from the hTERT (human telomerase reverse transcriptase) gene promoter in the fully open. Angiogenesis is the key processes of tumor survival and metastasis. Targeted inhibition of tumor angiogenesis may indirectly inhibit tumor growth and metastasis. HCC is a highly vascularized tumor,therefore, using its specific hTERT promoter-driven anti-angiogenic genes to inhibit liver angiogenesis is a promising therapeutic strategy.
Objective
Construct the plasmid of phTERT-tumstatin gene vector driven by the hTERT promoter , To observe the specific expression and secretion of tumstatin transduced into HepG2 ,driven by the hTERT promoter and its anti-angiogenic effect in vitro.
Methods
1.Cells HepG2、L-02 and HUVEC were cultured in vitro cell culture. 2.Constructed phTERT-tumstatin plasmid and its control group. 3.We transfected HepG2 and L-02 with the plasmid of phTERT-tumstatin. 4.The expression of EGFP was examined with fluorescence microscope observations;The secretion of target protein tumstatin from HepG2 cells was detected by Western blotting. 5.MTS assay was used to measure cell proliferation of HepG2 cells after stably transfected by plasmids. 6.The effect of conditioned mediun (containing target protein or not) on the proliferation of HUVEC cells was detected by MTS assay. 7.The effect of target protein Tumstatin on the formation of HUVEC celluar tube structure can be observed through counting the number of tube branches.
Results
1.We successfully constructed phTERT-tumstatin, pCMV-tumstatin (positive control) and phTERT-EGFP(negative control) plasmids. 2.The specific expression and secretion of tumstatin was only observed in HepG2, but not in normal liver L-02 cells. 3.The target protein contained in CM-T inhibited the proliferation of HUVEC cells, inhibition rate reached (56.49±0.33)%. 4.The number of tube branches on Matrigel-coated plates supplemented with conditioned medium CM-T was (3.33±1.53)%. Compared with conditioned medium CM-N and CM-NT, supplement of CM-T significantly inhibited the cellular tube structure formation of HUVEC cells on Matrigel-coated plates【(3.33±1.53)% vs (24.44±3.11)%、(23.94±2.92)%,P<0.01)】。
Conclusion
The plasmid of hTERT-tumstatin was successfully constructed. The target protein selective expression and secretion in liver cancer cells can inhibit the proliferation of endothelial cells and vascular structure formation and almost have no influence on normal hepatic cells. The hTERT-tumstatin has a potential effect of targeted inhibition of angiogenesis .
引文
1. Hamano Y, Zeisberg M, Sugimoto H, et al. Physiological levels of tumstatin, a fragment of collagen IV alpha3 chain, are generated by MMP-9 proteolysis and suppress angiogenesis via alphaV beta3 integrin[J]. Cancer Cell, 2003, 3(6):589-601.
2. Goto T, Ishikawa H, Matsumoto K,et al. Tum-1,a tumstatin fragment, gene delivery into hepatocellular carcinoma suppresses tumor growth through inhibiting angiogenesis[J]. Int J Oncol, 2008 , 33(1):33-40
3. Zhang X, Xu W, Qian H, et al. Mesenchymal stem cells modified to express lentivirus-TNFalpha-Tumstatin45-132 inhibit the growth of prostate cancer[J]. J Cell Mol Med, 2011, 15(2)433-444
4. Ohsako Y, Tsutsumi K, Li X, et al. Studies on RNA-protein interactions and enzyme activity by mutational analysis of telomerase[J]. Nucleic Acids Symp Ser, 2007,(51):419-420
5.Song JS, Kim HP. Adenovirus-mediated HSV-TK gene therapy using the human telomerase promoter induced apoptosis of small cell lung cancer cell line[J].Oncol Rep,2004, 12(2):443-447.
6. Nemunaitis J, Tong AW, Nemunaitis M, et al. A phase I study of telomerase-specific replication competent oncolytic adenovirus (telomelysin) for various solid tumors[J]. Mol Ther, 2010 , 18(2):429-434
7.Miyoshi T, Hirohata S, Ogawa H,et al. Tumor-specific expression of the RGD-a3(IV)NC1 domain suppresses endothelial tube formation and tumor growth in mice[J]. FASEB J,2006 20(11);1904-1906
8. Ribeiro-Silva A, Becker de Moura H, Ribeiro do Vale F, et al. The differential regulation of human telomerase reverse transcriptase and vascular endothelial growth factor may contribute to the clinically more aggressive behavior of p693-positive breast carcinomas[J]. Int J Biol Markers, 2005, 20(4):227-234
9. Bruix J, Sherman M. Management of hepatocellular carcinoma: an update[J]. Hepatology, 2011 , 53(3):1020-1022
10. Boosani CS, Varma AK, Sudhakar A. Validation of Different Systems for Tumstatin Expression and its in-vitro and iv-vivo Activities[J]. J Cancer Sci Ther, 2010 , 2009:8-18
11. Boosani CS, Mannam AP, Cosgrove D, et al. Regulation of COX-2 mediated signaling by alpha3 type IV noncollagenous domain in tumor angiogenesis[J].Blood , 2007, 110(4):1168–1177.
12. Sudhakar A, Sugimoto H, Yang C, et al. Human tumstatin and human endostatin exhibit distinct antiangiogenic activities mediated by alpha v beta 3 and alpha 5 beta 1 integrins[J]. Proc Natl Acad Sci USA, 2003, 100(8):4766–4771.
13.Hamano Y,Kalluri R. Tumstatin, the NC1 domain of alpha3 chain of type IV collagen, is an endogenous inhibitor of pathological angiogenesis and suppresses tumor growth[J]. Biochem Biophys Res Commun , 2005 , 333(2): 292-298
14. Yang YP, Xu CX, Hou GS, et al. Effects of eukaryotic expression plasmid encoding human tumstatin gene on endothelial cells in vitro[J]. Chin MedJ(Engl), 2010 , 123(16):2269-2273.
15. Zhang X, Xu W, Qian H, et al. Mesenchymal stem cells modified to express lentivirus-TNFalpha-Tumstatin45-132 inhibit the growth of prostate cancer[J]. J Cell Mol Med, 2011, 15(2):433-444
16. Persano L, Crescenzi M, Indraccolo S. Anti-angiogenic gene therapy of cancer:Current status and future prospects[J]. Mol Aspects Med, 2007 , 28(1):87-114
17. Indran IR, Hande MP, Pervaiz S. Tumor cell redox state and mitochondria at the center of the non-canonical activity of telomerase reverse transcriptase[J]. Mol Aspects Med, 2010 ,31(1):21-28.
18. Yang S, Wang C, Fang X, et al. Fusion of C3d molecule with neutralization epitope(s) of hepatitis E virus enhances antibody avidity maturation and neutralizing activity following DNA immunization[J]. Virus Res, 2010 , 151(2):162-169.
19. Luo M, Tao P, Li J, et al. Immunization with plasmid DNA encoding influenza A virus nucleoprotein fused to a tissue plasminogen activator signal sequence elicits strong immune responses and protection against H5N1 challenge in mice[J]. J Virol Methods, 2008 ,154(1-2):121-127.
20. Maeshima Y, Colorado PC, Torre A, et al. Distinct antitumor properties of a type IV collagen domain derived from basement membrane[J]. J Biol Chem , 2000 , 275(28):21340–21348.
21. Kawaguchi T, Yamashita Y,Kanamori M, et al. The PTEN/Akt pathway dictates the direct alphaVbeta3-dependent growth-inhibitory action of an active fragment of tumstatin in glioma cells in vitro and in vivo[J]. Cancer Res , 2006, 66(23): 11331-11340
22. Mayoral R, Fernandez-Martinez A, Bosca L, et al. Prostaglandin E2 promotes migration and adhesion in hepatocellular carcinoma cells[J]. Carcinogenesis, 2005, 26(4): 753-761
23. Peyrou M, Bourgoin L, Foti M. PTEN in liver diseases and cancer [J]. World J Gastroenterol, 2010 , 16(37): 4627-4633
[1] Zhao Y, Wang S, Popova EY, et al. Rearrangement of upstream sequences of the hTERT gene during cellular immortalization[J]. Genes Chromosomes Cancer,2009 ,48(11):963-974.
[2] Li NF, Kocher HM, Salako MA, et al. A novel function of colony-stimulating factor 1 receptor in hTERT immortalization of human epithelial cells[J]. Oncogene, 2009, 28(5):773-80.
[3] Cassar L, Nicholls C, Pinto AR, et al. Bone morphogenetic protein-7 induces telomerase inhibition, telomere shortening, breast cancer cell senescence, and death via Smad3[J]. FASEB J,2009 ,23(6):1880-1892.
[4] Str(?)t K, Liu C, Rahbar A, et al. Activation of telomerase by human cytomegalovirus[J]. J Natl Cancer Inst,2009 , 101(7):488-497.
[5] Bilsland AE, Hoare S, Stevenson K et al. Dynamic telomerase gene suppression via network effects of GSK3 inhibition[J]. PLoS One,2009 ,4(7) :e6459.
[6] Lacerte A, Korah J, Roy M, et al. Transforming growth factor-beta inhibits telomerase through SMAD3 and E2F transcription factors[J]. Cell Signal,2008, 20: 50-59.
[7] Moriai M, Tsuji N, Kobayashi D ,et al. Down-regulation of hTERT expression plays an important role in 15-deoxy-Delta12,14-prostaglandin J2-induced apoptosis in cancer cells[J]. Int J Oncol,2009 , 34(5):1363-1372.
[8] Kyo S, Takakura M, Fujiwara T, et al. Understanding and exploiting hTERT promoter regulation for diagnosis and treatment of human cancers[J]. Cancer Sci, 2008, 99(8): 1528–1538.
[9] Choi J, Min N, Park J, et al. TSA-induced DNMT1 down-regulation represses hTERT expression via recruiting CTCF into demethylated core promoter region of hTERT in HCT116[J]. Biochem Biophys Res Commun,2010, 391(1): 449-454.
[10] Phipps SM, Love WK, White T ,et al. Retinoid-induced histone deacetylation inhibits telomerase activity in estrogen receptor-negative breast cancer cells[J]. Anticancer Res,2009 , 29(12):4959-4964.
[11] Kumari A, Srinivasan R, Vasishta RK, et al. Positive regulation of human telomerase reverse transcriptase gene expression and telomerase activity by DNA methylation in pancreatic cancer[J]. Ann Surg Oncol,2009 ,16(4):1051-1059.
[12] Iliopoulos D, Satra M, Drakaki A, et al. Epigenetic regulation of hTERT promoter in hepatocellular carcinomas[J]. Int J Oncol,2009 , 34(2):391-399.
[13] Kojima T, Watanabe Y, Hashimoto Y, et al. In vivo biological purging for lymph node metastasis of human colorectal cancer by telomerase-specific oncolytic virotherapy[J]. Ann Surg,2010 , 251(6):1079-1086.
[14] Takakura M, Nakamura M, Kyo S , et al. Intraperitoneal administration oftelomerase-specific oncolytic adenovirus sensitizes ovarian cancer cells to cisplatin and affects survival in a xenograft model with peritoneal dissemination[J]. Cancer Gene Ther,2010, 17(1):11-19.
[15] Huang P, Kaku H, Chen J, et al. Potent antitumor effects of combined therapy with a telomerase-specific, replication-competent adenovirus (OBP-301) and IL-2 in a mouse model of renal cell carcinoma[J]. Cancer Gene Ther,2010 , 17(7):484-491.
[16] Kumari A, Srinivasan R, Wig JD. Effect of c-MYC and E2F1 gene silencing and of 5-azacytidine treatment on telomerase activity in pancreatic cancer-derived cell lines[J]. Pancreatology,2009, 9(4):360-368.
[17] Hu Z, Robbins JS, Pister A, et al. A modified hTERT promoter-directed oncolytic adenovirus replication with concurrent inhibition of TGFbeta signaling for breast cancer therapy[J]. Cancer Gene Ther,2010 ,17(4):235-243.
[18] Song MS, Jeong JS, Ban G, et al. Validation of tissue-specific promoter-driven tumor-targeting trans-splicing ribozyme system as a multifunctional cancer gene therapy device in vivo[J]. Cancer Gene Ther,2009 ,16(2):113-125.
2. Goto T, Ishikawa H, Matsumoto K,et al. Tum-1,a tumstatin fragment, gene delivery into hepatocellular carcinoma suppresses tumor growth through inhibiting angiogenesis[J]. Int J Oncol, 2008 , 33(1):33-40
3. Zhang X, Xu W, Qian H, et al. Mesenchymal stem cells modified to express lentivirus-TNFalpha-Tumstatin45-132 inhibit the growth of prostate cancer[J]. J Cell Mol Med, 2011, 15(2)433-444
4. Ohsako Y, Tsutsumi K, Li X, et al. Studies on RNA-protein interactions and enzyme activity by mutational analysis of telomerase[J]. Nucleic Acids Symp Ser, 2007,(51):419-420
5.Song JS, Kim HP. Adenovirus-mediated HSV-TK gene therapy using the human telomerase promoter induced apoptosis of small cell lung cancer cell line[J].Oncol Rep,2004, 12(2):443-447.
6. Nemunaitis J, Tong AW, Nemunaitis M, et al. A phase I study of telomerase-specific replication competent oncolytic adenovirus (telomelysin) for various solid tumors[J]. Mol Ther, 2010 , 18(2):429-434
7.Miyoshi T, Hirohata S, Ogawa H,et al. Tumor-specific expression of the RGD-a3(IV)NC1 domain suppresses endothelial tube formation and tumor growth in mice[J]. FASEB J,2006 20(11);1904-1906
8. Ribeiro-Silva A, Becker de Moura H, Ribeiro do Vale F, et al. The differential regulation of human telomerase reverse transcriptase and vascular endothelial growth factor may contribute to the clinically more aggressive behavior of p693-positive breast carcinomas[J]. Int J Biol Markers, 2005, 20(4):227-234
9. Bruix J, Sherman M. Management of hepatocellular carcinoma: an update[J]. Hepatology, 2011 , 53(3):1020-1022
10. Boosani CS, Varma AK, Sudhakar A. Validation of Different Systems for Tumstatin Expression and its in-vitro and iv-vivo Activities[J]. J Cancer Sci Ther, 2010 , 2009:8-18
11. Boosani CS, Mannam AP, Cosgrove D, et al. Regulation of COX-2 mediated signaling by alpha3 type IV noncollagenous domain in tumor angiogenesis[J].Blood , 2007, 110(4):1168–1177.
12. Sudhakar A, Sugimoto H, Yang C, et al. Human tumstatin and human endostatin exhibit distinct antiangiogenic activities mediated by alpha v beta 3 and alpha 5 beta 1 integrins[J]. Proc Natl Acad Sci USA, 2003, 100(8):4766–4771.
13.Hamano Y,Kalluri R. Tumstatin, the NC1 domain of alpha3 chain of type IV collagen, is an endogenous inhibitor of pathological angiogenesis and suppresses tumor growth[J]. Biochem Biophys Res Commun , 2005 , 333(2): 292-298
14. Yang YP, Xu CX, Hou GS, et al. Effects of eukaryotic expression plasmid encoding human tumstatin gene on endothelial cells in vitro[J]. Chin MedJ(Engl), 2010 , 123(16):2269-2273.
15. Zhang X, Xu W, Qian H, et al. Mesenchymal stem cells modified to express lentivirus-TNFalpha-Tumstatin45-132 inhibit the growth of prostate cancer[J]. J Cell Mol Med, 2011, 15(2):433-444
16. Persano L, Crescenzi M, Indraccolo S. Anti-angiogenic gene therapy of cancer:Current status and future prospects[J]. Mol Aspects Med, 2007 , 28(1):87-114
17. Indran IR, Hande MP, Pervaiz S. Tumor cell redox state and mitochondria at the center of the non-canonical activity of telomerase reverse transcriptase[J]. Mol Aspects Med, 2010 ,31(1):21-28.
18. Yang S, Wang C, Fang X, et al. Fusion of C3d molecule with neutralization epitope(s) of hepatitis E virus enhances antibody avidity maturation and neutralizing activity following DNA immunization[J]. Virus Res, 2010 , 151(2):162-169.
19. Luo M, Tao P, Li J, et al. Immunization with plasmid DNA encoding influenza A virus nucleoprotein fused to a tissue plasminogen activator signal sequence elicits strong immune responses and protection against H5N1 challenge in mice[J]. J Virol Methods, 2008 ,154(1-2):121-127.
20. Maeshima Y, Colorado PC, Torre A, et al. Distinct antitumor properties of a type IV collagen domain derived from basement membrane[J]. J Biol Chem , 2000 , 275(28):21340–21348.
21. Kawaguchi T, Yamashita Y,Kanamori M, et al. The PTEN/Akt pathway dictates the direct alphaVbeta3-dependent growth-inhibitory action of an active fragment of tumstatin in glioma cells in vitro and in vivo[J]. Cancer Res , 2006, 66(23): 11331-11340
22. Mayoral R, Fernandez-Martinez A, Bosca L, et al. Prostaglandin E2 promotes migration and adhesion in hepatocellular carcinoma cells[J]. Carcinogenesis, 2005, 26(4): 753-761
23. Peyrou M, Bourgoin L, Foti M. PTEN in liver diseases and cancer [J]. World J Gastroenterol, 2010 , 16(37): 4627-4633
[1] Zhao Y, Wang S, Popova EY, et al. Rearrangement of upstream sequences of the hTERT gene during cellular immortalization[J]. Genes Chromosomes Cancer,2009 ,48(11):963-974.
[2] Li NF, Kocher HM, Salako MA, et al. A novel function of colony-stimulating factor 1 receptor in hTERT immortalization of human epithelial cells[J]. Oncogene, 2009, 28(5):773-80.
[3] Cassar L, Nicholls C, Pinto AR, et al. Bone morphogenetic protein-7 induces telomerase inhibition, telomere shortening, breast cancer cell senescence, and death via Smad3[J]. FASEB J,2009 ,23(6):1880-1892.
[4] Str(?)t K, Liu C, Rahbar A, et al. Activation of telomerase by human cytomegalovirus[J]. J Natl Cancer Inst,2009 , 101(7):488-497.
[5] Bilsland AE, Hoare S, Stevenson K et al. Dynamic telomerase gene suppression via network effects of GSK3 inhibition[J]. PLoS One,2009 ,4(7) :e6459.
[6] Lacerte A, Korah J, Roy M, et al. Transforming growth factor-beta inhibits telomerase through SMAD3 and E2F transcription factors[J]. Cell Signal,2008, 20: 50-59.
[7] Moriai M, Tsuji N, Kobayashi D ,et al. Down-regulation of hTERT expression plays an important role in 15-deoxy-Delta12,14-prostaglandin J2-induced apoptosis in cancer cells[J]. Int J Oncol,2009 , 34(5):1363-1372.
[8] Kyo S, Takakura M, Fujiwara T, et al. Understanding and exploiting hTERT promoter regulation for diagnosis and treatment of human cancers[J]. Cancer Sci, 2008, 99(8): 1528–1538.
[9] Choi J, Min N, Park J, et al. TSA-induced DNMT1 down-regulation represses hTERT expression via recruiting CTCF into demethylated core promoter region of hTERT in HCT116[J]. Biochem Biophys Res Commun,2010, 391(1): 449-454.
[10] Phipps SM, Love WK, White T ,et al. Retinoid-induced histone deacetylation inhibits telomerase activity in estrogen receptor-negative breast cancer cells[J]. Anticancer Res,2009 , 29(12):4959-4964.
[11] Kumari A, Srinivasan R, Vasishta RK, et al. Positive regulation of human telomerase reverse transcriptase gene expression and telomerase activity by DNA methylation in pancreatic cancer[J]. Ann Surg Oncol,2009 ,16(4):1051-1059.
[12] Iliopoulos D, Satra M, Drakaki A, et al. Epigenetic regulation of hTERT promoter in hepatocellular carcinomas[J]. Int J Oncol,2009 , 34(2):391-399.
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