减毒沙门菌作为新型肿瘤基因治疗载体的构建及其有效性检测
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摘要
肿瘤的基因治疗即是将目的基因用基因技术导入靶细胞,并使其表达、执行或介导对肿瘤的杀伤和抑制作用,从而达到治疗之目的。选择合适的载体一直是肿瘤基因治疗研究中的重点。鼠伤寒沙门菌是一种兼性厌氧菌,它可以在乏氧的肿瘤组织内聚集和繁殖,且聚集在肿瘤部位(包括转移灶)的数量是正常组织的1000倍。它与宿主之间的反应通过Ⅲ型分泌机制介导,这种机制使细菌的效应蛋白能直接转移到真核细胞发挥作用,使其可以传递表达效应基因并同时表达多种治疗性蛋白。以上两种特性为沙门菌用作肿瘤基因治疗的靶向载体提供了可能。而近年来迅速发展的基因工程技术能将沙门菌的致病基因敲除后得到减毒菌株,为安全性提供了保证。
本实验采用细菌导向的前药酶疗法,利用沙门菌选择性定植于肿瘤组织从而将前药活化酶基因靶向转运至肿瘤组织内表达,通过全身运用无毒性的前药,使前药仅能在肿瘤组织内被活化成具有抗癌活性的药物,从而达到杀死肿瘤细胞并减少抗癌活性药物对正常组织的影响。
第一部分的主要内容为利用减毒沙门菌VNP20009构建新型载体ST-CD菌株。首先通过Red同源重组技术,获得了营养缺陷菌株ST/Δasd,然后构建pAsd-CD质粒,将质粒导入营养缺陷菌株ST/Δasd,最后成功筛选并鉴定出新型载体ST-CD菌株。经检测ST-CD菌株可将外源性5-氟胞嘧啶转化为5-氟尿嘧啶。
第二部分的主要内容为检测新型载体ST-CD菌株的低毒性和靶向性。分别将ST-CD菌株和野生沙门菌自小鼠尾静脉注入体内,发现接受ST-CD的小鼠没有死亡,而接受野生沙门菌的小鼠4天后全部死亡；将ST-CD注入MFC小鼠体内,发现第6天时肿瘤/肝脏的菌量比约为1020：l。说明新型载体ST-CD具有良好的低毒性和肿瘤靶向性。
第三部分的主要内容为将新型载体ST-CD注入MFC小鼠体内并在小鼠腹腔内注射5-氟胞嘧啶,观察肿瘤生长情况。结果发现和对照组相比,ST-CD/5-Fc系统有着良好的抑瘤效果,其抑制肿瘤生长的原因可能与ERK表达受抑制有关。
Cancer gene therapy is to use gene technology translating target genes into target cells and expression, execution or mediated tumor destruction and inhibition, so as to achieve the goal of treatment. Choose the right carrier has been the focus of the cancer gene therapy research. Salmonella typhimurium is a facultative anaerobic bacteria, it can accumulate in hypoxic tumor tissue and reproduction, and gathered at the tumor site (including metastases)1,000times than the normal tissue. Between it and the host mediated by the type III secretion mechanism, this mechanism of bacterial effector proteins directly transferred to the eukaryotic cells, so that it can dilivery the expression of the effects of genes and expression of a variety of therapeutic proteins. The above two characteristics of Salmonella make it possible to be a targeting vector for cancer gene therapy. Attenuated Salmonella strains can be made by knockout virulence gene with the rapid development of genetic engineering techniques in recent years.
In this study, we choose bacteria-directed enzyme prodrug therapy, so the activating prodrug enzyme gene can be transferded to the tumor and be expressed by the selective colonization of Salmonella in tumor tissues. Though the systemic use of non-toxic prodrug, the prodrug can only be activated within the tumor tissue with anti-cancer activity of drugs, so as to achieve the killing of tumor cells and reduce the impact of anti-cancer drugs on normal tissues.
The first part focused on the construction of the new vector ST-CD strains by using attenuated Salmonella typhimurium VNP20009. First, we get auxotrophic strains ST/Aasd by the Red homologous recombination technology, then we construct Asd-CD plasmid and introduce it into the auxotrophics strains ST/Aasd, and at last we sucessly screen and identify the new vector of ST-CD strain. We find that the new vector ST-CD can convert exogenous5-fluorocytosine into5-fluorouracil.
The second part focused on the hypotoxicity and tumor-targeting ability of the new vector ST-CD. Non-tumor-bearing mice were infected intravenously with ST-CD or wild-type. All mice infected with wild-type S.typhymurium expired by4days. In contrast, all mice infected with ST-CD survived. Then tumor-bearing mice were infected intravenously with ST-CD, the tumor/liver bacteria ratios were1020:1by day6after infection. It seems the new vector ST-CD has the very ability of tumor-targeting and hypotoxicity.
The third part focused on the observation of tumor bearing mice by infection intravenously with ST-CD and intraperitoneal injection of5-fluorocytosine. The results showed that compared to the control group, ST-CD/5-Fc system has good anti-tumor effects which may be affected by the inhibition of ERK expression.
本实验采用细菌导向的前药酶疗法,利用沙门菌选择性定植于肿瘤组织从而将前药活化酶基因靶向转运至肿瘤组织内表达,通过全身运用无毒性的前药,使前药仅能在肿瘤组织内被活化成具有抗癌活性的药物,从而达到杀死肿瘤细胞并减少抗癌活性药物对正常组织的影响。
第一部分的主要内容为利用减毒沙门菌VNP20009构建新型载体ST-CD菌株。首先通过Red同源重组技术,获得了营养缺陷菌株ST/Δasd,然后构建pAsd-CD质粒,将质粒导入营养缺陷菌株ST/Δasd,最后成功筛选并鉴定出新型载体ST-CD菌株。经检测ST-CD菌株可将外源性5-氟胞嘧啶转化为5-氟尿嘧啶。
第二部分的主要内容为检测新型载体ST-CD菌株的低毒性和靶向性。分别将ST-CD菌株和野生沙门菌自小鼠尾静脉注入体内,发现接受ST-CD的小鼠没有死亡,而接受野生沙门菌的小鼠4天后全部死亡；将ST-CD注入MFC小鼠体内,发现第6天时肿瘤/肝脏的菌量比约为1020：l。说明新型载体ST-CD具有良好的低毒性和肿瘤靶向性。
第三部分的主要内容为将新型载体ST-CD注入MFC小鼠体内并在小鼠腹腔内注射5-氟胞嘧啶,观察肿瘤生长情况。结果发现和对照组相比,ST-CD/5-Fc系统有着良好的抑瘤效果,其抑制肿瘤生长的原因可能与ERK表达受抑制有关。
Cancer gene therapy is to use gene technology translating target genes into target cells and expression, execution or mediated tumor destruction and inhibition, so as to achieve the goal of treatment. Choose the right carrier has been the focus of the cancer gene therapy research. Salmonella typhimurium is a facultative anaerobic bacteria, it can accumulate in hypoxic tumor tissue and reproduction, and gathered at the tumor site (including metastases)1,000times than the normal tissue. Between it and the host mediated by the type III secretion mechanism, this mechanism of bacterial effector proteins directly transferred to the eukaryotic cells, so that it can dilivery the expression of the effects of genes and expression of a variety of therapeutic proteins. The above two characteristics of Salmonella make it possible to be a targeting vector for cancer gene therapy. Attenuated Salmonella strains can be made by knockout virulence gene with the rapid development of genetic engineering techniques in recent years.
In this study, we choose bacteria-directed enzyme prodrug therapy, so the activating prodrug enzyme gene can be transferded to the tumor and be expressed by the selective colonization of Salmonella in tumor tissues. Though the systemic use of non-toxic prodrug, the prodrug can only be activated within the tumor tissue with anti-cancer activity of drugs, so as to achieve the killing of tumor cells and reduce the impact of anti-cancer drugs on normal tissues.
The first part focused on the construction of the new vector ST-CD strains by using attenuated Salmonella typhimurium VNP20009. First, we get auxotrophic strains ST/Aasd by the Red homologous recombination technology, then we construct Asd-CD plasmid and introduce it into the auxotrophics strains ST/Aasd, and at last we sucessly screen and identify the new vector of ST-CD strain. We find that the new vector ST-CD can convert exogenous5-fluorocytosine into5-fluorouracil.
The second part focused on the hypotoxicity and tumor-targeting ability of the new vector ST-CD. Non-tumor-bearing mice were infected intravenously with ST-CD or wild-type. All mice infected with wild-type S.typhymurium expired by4days. In contrast, all mice infected with ST-CD survived. Then tumor-bearing mice were infected intravenously with ST-CD, the tumor/liver bacteria ratios were1020:1by day6after infection. It seems the new vector ST-CD has the very ability of tumor-targeting and hypotoxicity.
The third part focused on the observation of tumor bearing mice by infection intravenously with ST-CD and intraperitoneal injection of5-fluorocytosine. The results showed that compared to the control group, ST-CD/5-Fc system has good anti-tumor effects which may be affected by the inhibition of ERK expression.
引文
[1]Figueroa-Bossi N,Uzzau S,Malorial D,et al.Variable assortment of prophages provides a transferable repertoire of pathogenic determinants in salmonella. Mol Microbial,2001,39(2):260-71.
[2]Platt J,Sodi S,Kelley M,et al. Antitumour effects of genetically engineered Salmonella in combination with radiation. Eur J Cancer,2000,36(18):2397-42.
[3]齐桓,李玉华,郑少斌等.以沙门氏菌为载体的口服基因治疗对小鼠肿瘤的作用[J].南方医科大学学报,2006,26(12),1738-1741
[4]Low KB, Ittensohn m, Le T,et al. Lipid A mutant Salmonella with suppressed virulence and TNFalpha induction retain tumor-targeting in vivo. Nat Biotechnol,1999,17(1):37-41.
[5]Sonerville JE Jr, Cassiano L, Darveau RP. Escherichia Coli msbB gene as virulence factor and a therapeutic target. Infect lmmun,1999,67(12):6583-90.
[6]Low KB, Ittensohn M, Luo X, et al Construction of VNP2009:a novel, genetically stable antibiotic-sensitive strain of tumor-targeting Salmonella for parenteral administration in humans. Methods mol Med,2004,90:47-60.
[7]Pawelek JM, Low KB. Bermudes D. Bacteria as tumour-targeting vectors. Lancet Oncol,2003,4(9):548-556.
[8]Clairmont C,Lee K,Pike J,et al. Biodistribution and Genetic Stability of the Novel Antitumor Agent VNP20009,a Genetically Modified Strain of Salmonella typhimurium.J Infect Dis,2000,181(6):19962-20021
[9]Nakamura H, MuLlen JT, Chandrasekhar S, Pawlik TM, Yoon SS,Tanabe KK. Multimodality therapy with a replication-conditional herpes simplex virus 1 mutant that expresses yeast cytosinedeaminase for intratumoral conversion of 5-fluorocytosine to 5-fluorouracil. Cancer Res 2001; 61:5447-5452
[10]Schleifer KH, Kandler O. Peptidoglycan types of bacterial cell walls and their taxonomic implication[J]. Bacterial Rev,1972,36(4):407
[11]Nakayama K, Kelly SM, Curtiss IIIR, Construction of an Asd+ expression-cloning vector:stable maintenance and high level expression of cloned genes in a Salmonella vaccine strain. Biotechnology,1988,6:693-697
[12]Carter D M, Radding CM. The role of exonuclease and β protein of phage λ in genetic recombination substrate specificity and the mode of action of lambda exonuclease. J Biol Chem,1971,246(8):2502-2512
[13]Kovall R, Matthews B W. Toroidal structure of lambda exonuclease. Science,1997,277 (5333):1824-1827
[14]Muniyappa K, Radding C M. The homologous recombination system of phage λ. Pairing activities of β protein. J Biol Chem,1986,261 (16):7472-7478
[15]Datsenko K A,Wanner B L. One step inactivation of chromosomal genes in Escherichia coli K212 using PCR products. Proc Natl Acad Sci USA,2000,97 (12):6640-6645
[16]王恒,冯尔玲,史兆兴等.用Red系统快速敲除痢疾杆菌asd基因.军事医学科学院院刊,2002,26(3)：161-164
[17]腾家波载体-宿主平衡致死系统在减毒伤寒沙门氏活菌苗中的应用.国外医学免疫学分册,1999,22(3)：143-146
[1]Yamamoto M, Jiang P, Yang M, et al Cellular Dynamics Visualized in Live Cells in vitro and in vivo by Differential Dual-Color Nuclear-Cytoplasmic Fluorescent-Protein Expression [J], Cancer Res,2004,64(8):4251-4256.
[2]Figueroa BN, Uzzau S, Maloriol D, et al. Variable assortment of prophages provides a transferable repertoire of pathogenic determinants in Salmonella [J] Mol Microbiol,2001,39(2):260-271.
[3]Inoue M, Mukai M, Hamanaka Y, et al. Targeting hypoxic cancer cells with a protein prodrug is effective in experimental malignant ascites[J]. Int J Oncol,2004,25(3):713-720.
[4]Clairmont C, Lee K, Pike J, et al. Bio-distribution and genetic Stability of the novel anti-tumor agent VNP20009, a genetically modified strain of Salmonella typhimurium[J].J Infect Dis,2000,181(6):1996-2000.
[5]Cunningham C, Nemunaitis J. A phase I trial of genetically modified Salmonella typhimurium expressing cytosine deaminase (TAPET-CD, VNP20029) administered by intratumoral injection in combination with 5-fluorocytosine for patients with advanced or metastatic cancer[J]. Hum Gene Ther,2001,12(12):1594-1596.
16] Wei MQ, Ellem KA, Dunn P, et al. Facultative or obligate anaerobic bacteria have the potential for multimodality therapy of solid tumors[J]. Eur J Cancer,2007,43(3):490-496
[7]Sznol M, Lin SL, Bermudes D, et al. Use of preferentially replicating bacteria for the treatment of cancer[J]. J Clin invest,2000,105(8):1027-1030.
[8]Theys J, Barbe S, Landuyt W, et al Tumor-specific gene delivery using genetically engineered bacteria[J]. Curr Gene Ther,2003,3(3):207-221.
[1]KiyokawaE, TakaiS, TanakaM, etal.Over expression of ERK, anEPH family receptor protein tyrosine kinase, in various human tumors[J].Cancer Res,1994,54(14):3645-3650
[2]IwaseT, TanakaM, SuzukiM, et al.Identification of protein-tyrosine kinase genes preferentially expressed in embryo stomach and gastric cancer. Biochem Biophys Res Commun,1993,194(2):698-705.
[3]BangYJ, KwonJH, KangSH, et al. Inereased MAPK aetivity and MKP-1 overexpression in human gastric adenocarcinoma. Biochem Biophys Res Commun,1998,250(1):43-47.
[4]Parker RC, Plummer HC, Siebenmann CO, et al. Effect of histolyticus infection and toxin on transplantable mouse tumors. Proc Soc Exp Biol Med,1947,66(2):461-7
[5]Zh ao M, Yang M, Ma H, et al. Targeted Therapy with a Salmonell a Typhimurium Leucine-Arginine Auxotroph Cures Orthotopic Human Breast Tumors in Nude Mice[J]. Cancer Res,2006,66 (15):7647-7652.
[6]Sorenson BS, Banton KL, Frykman NL, et al. Attenuated Salmonella typhimurium with IL-2 gene reduces pulmonary metastases in murine osteosarcoma. Clin Orthop Relat Res,2008,466(6):1285-91
[8]Zhang L, Gao LF, Zhao LJ, et al. Intratumoral delivery and suppression of prostate tumor growth by attenuate Sdalmonella enterica serovar typhimuriumc arrying plasmid-based small interfering RNAs. Cancer Res,2007, 67(12):5859-64
[9]King I, Bermudes D, Lin S, et al. Tumor-targeted Salmonella expressing cytosine deaminase as an anticancer agent. HumGene Ther,2002,13(10):1225-33
[10]Seger R, Krebs EG.The MAPK signaling caseade. FASEB J,1995,9(9):726-735
[11]Alejandro EU, Johnson JD. Inhibition of Raf-1 alters multiple downstream pathways to induce pancreatic beta-cell apoptosis [J] J BiolChem,2008,283(4):2407-2417.
[12]Howie HL, Shiflett SL, So M. ExtracelljiLar signal-reguLated kmase activation by Neisseria gonorrhoeae downregulates epithelial cell proapoptotic proteins Bad and Bim [J].Infect Immun,2008,76(6):2715-2721.
1 Nauts, HC, Swift, WE, and Coley, BL. The treatment of malignant tumours by bacterial toxins as developed by the late William B. Coley, MD. Cancer Res,1946, 38:9.
2 Eisentein TK, Bushnell B, Meissler JJ. Immunotherapy of a plasmacytoma with attenuated Salmonella. Med Oncol,1995,12(2):103-108.
3 Pawelek, J, Low, KB, and Bermudes. Tumor-targeted Salmonella as a novel anti-cancer vector. Cancer Res,1997,57:4537.
4 Kochi SK, Killeen KP, Pyan US, et al. Advances in the development of bacterial vector technology. Expert Rev Vaccine,2003,2(1):31-43.
5 Bermudes D, Zheng LM, King IC, Live bacteria as anticancer agents and tumor-selective protein delivery vectors. Curr Opion Drug Discov Devel,2002,5(2): 194-9.
6 Nishikawa H, Sato E, Briones G,et al. In vivo antigen delivery by a Salmonella typhimurium type Ⅲ secretion system for therapeutic cancer vaccines. J Clin Invest,2006,116:1946-1954.
7 Niethammer AG, Primus FJ, Xiang R, et al. An oral DNA vaccine against human carcinoembryonic antigen (CEA) prevents growth and dissemination of Lewis lung carcinoma in CEA transgenic mice.Vaccine,2001,20(3-4):421-429.
8 Low KB, Ittensohn M, Luo X,et al. Construction of VNP20009:a novel, genetically stable antibiotic-sensitive strain of tumor-targeting Salmonella for parenteral administration in humans. Methods Mol Med,2004,90:47-60.
9 Al-Ramadi BK, Fernandez-Cabezudo MJ, El-Hasasna H, et al. Potent anti-tumor activity of systemically-administered IL2-expressing Salmonella correlates with decreased angiogenesis and enhanced tumor apoptosis. Clinical Immunology, 2009,130:89-97.
10 Sorenson BS, Banton KL, Frykman NL,et al. Attenuated Salmonella typhim-urium with IL-2 gene reduces puLmonary metastases in murine osteosarcoma. Clin Orthop Relat Res,2008,466(6):1285-1291.
11 Bereta M, Hayhurst A, Gajda M, et al.Improving tumor targeting and therap-eutic potential of Salmonella VNP20009 by displaying cell surface CEA-specific antibodies.Vaccine,2007,25:4183-4192.
[2]Platt J,Sodi S,Kelley M,et al. Antitumour effects of genetically engineered Salmonella in combination with radiation. Eur J Cancer,2000,36(18):2397-42.
[3]齐桓,李玉华,郑少斌等.以沙门氏菌为载体的口服基因治疗对小鼠肿瘤的作用[J].南方医科大学学报,2006,26(12),1738-1741
[4]Low KB, Ittensohn m, Le T,et al. Lipid A mutant Salmonella with suppressed virulence and TNFalpha induction retain tumor-targeting in vivo. Nat Biotechnol,1999,17(1):37-41.
[5]Sonerville JE Jr, Cassiano L, Darveau RP. Escherichia Coli msbB gene as virulence factor and a therapeutic target. Infect lmmun,1999,67(12):6583-90.
[6]Low KB, Ittensohn M, Luo X, et al Construction of VNP2009:a novel, genetically stable antibiotic-sensitive strain of tumor-targeting Salmonella for parenteral administration in humans. Methods mol Med,2004,90:47-60.
[7]Pawelek JM, Low KB. Bermudes D. Bacteria as tumour-targeting vectors. Lancet Oncol,2003,4(9):548-556.
[8]Clairmont C,Lee K,Pike J,et al. Biodistribution and Genetic Stability of the Novel Antitumor Agent VNP20009,a Genetically Modified Strain of Salmonella typhimurium.J Infect Dis,2000,181(6):19962-20021
[9]Nakamura H, MuLlen JT, Chandrasekhar S, Pawlik TM, Yoon SS,Tanabe KK. Multimodality therapy with a replication-conditional herpes simplex virus 1 mutant that expresses yeast cytosinedeaminase for intratumoral conversion of 5-fluorocytosine to 5-fluorouracil. Cancer Res 2001; 61:5447-5452
[10]Schleifer KH, Kandler O. Peptidoglycan types of bacterial cell walls and their taxonomic implication[J]. Bacterial Rev,1972,36(4):407
[11]Nakayama K, Kelly SM, Curtiss IIIR, Construction of an Asd+ expression-cloning vector:stable maintenance and high level expression of cloned genes in a Salmonella vaccine strain. Biotechnology,1988,6:693-697
[12]Carter D M, Radding CM. The role of exonuclease and β protein of phage λ in genetic recombination substrate specificity and the mode of action of lambda exonuclease. J Biol Chem,1971,246(8):2502-2512
[13]Kovall R, Matthews B W. Toroidal structure of lambda exonuclease. Science,1997,277 (5333):1824-1827
[14]Muniyappa K, Radding C M. The homologous recombination system of phage λ. Pairing activities of β protein. J Biol Chem,1986,261 (16):7472-7478
[15]Datsenko K A,Wanner B L. One step inactivation of chromosomal genes in Escherichia coli K212 using PCR products. Proc Natl Acad Sci USA,2000,97 (12):6640-6645
[16]王恒,冯尔玲,史兆兴等.用Red系统快速敲除痢疾杆菌asd基因.军事医学科学院院刊,2002,26(3)：161-164
[17]腾家波载体-宿主平衡致死系统在减毒伤寒沙门氏活菌苗中的应用.国外医学免疫学分册,1999,22(3)：143-146
[1]Yamamoto M, Jiang P, Yang M, et al Cellular Dynamics Visualized in Live Cells in vitro and in vivo by Differential Dual-Color Nuclear-Cytoplasmic Fluorescent-Protein Expression [J], Cancer Res,2004,64(8):4251-4256.
[2]Figueroa BN, Uzzau S, Maloriol D, et al. Variable assortment of prophages provides a transferable repertoire of pathogenic determinants in Salmonella [J] Mol Microbiol,2001,39(2):260-271.
[3]Inoue M, Mukai M, Hamanaka Y, et al. Targeting hypoxic cancer cells with a protein prodrug is effective in experimental malignant ascites[J]. Int J Oncol,2004,25(3):713-720.
[4]Clairmont C, Lee K, Pike J, et al. Bio-distribution and genetic Stability of the novel anti-tumor agent VNP20009, a genetically modified strain of Salmonella typhimurium[J].J Infect Dis,2000,181(6):1996-2000.
[5]Cunningham C, Nemunaitis J. A phase I trial of genetically modified Salmonella typhimurium expressing cytosine deaminase (TAPET-CD, VNP20029) administered by intratumoral injection in combination with 5-fluorocytosine for patients with advanced or metastatic cancer[J]. Hum Gene Ther,2001,12(12):1594-1596.
16] Wei MQ, Ellem KA, Dunn P, et al. Facultative or obligate anaerobic bacteria have the potential for multimodality therapy of solid tumors[J]. Eur J Cancer,2007,43(3):490-496
[7]Sznol M, Lin SL, Bermudes D, et al. Use of preferentially replicating bacteria for the treatment of cancer[J]. J Clin invest,2000,105(8):1027-1030.
[8]Theys J, Barbe S, Landuyt W, et al Tumor-specific gene delivery using genetically engineered bacteria[J]. Curr Gene Ther,2003,3(3):207-221.
[1]KiyokawaE, TakaiS, TanakaM, etal.Over expression of ERK, anEPH family receptor protein tyrosine kinase, in various human tumors[J].Cancer Res,1994,54(14):3645-3650
[2]IwaseT, TanakaM, SuzukiM, et al.Identification of protein-tyrosine kinase genes preferentially expressed in embryo stomach and gastric cancer. Biochem Biophys Res Commun,1993,194(2):698-705.
[3]BangYJ, KwonJH, KangSH, et al. Inereased MAPK aetivity and MKP-1 overexpression in human gastric adenocarcinoma. Biochem Biophys Res Commun,1998,250(1):43-47.
[4]Parker RC, Plummer HC, Siebenmann CO, et al. Effect of histolyticus infection and toxin on transplantable mouse tumors. Proc Soc Exp Biol Med,1947,66(2):461-7
[5]Zh ao M, Yang M, Ma H, et al. Targeted Therapy with a Salmonell a Typhimurium Leucine-Arginine Auxotroph Cures Orthotopic Human Breast Tumors in Nude Mice[J]. Cancer Res,2006,66 (15):7647-7652.
[6]Sorenson BS, Banton KL, Frykman NL, et al. Attenuated Salmonella typhimurium with IL-2 gene reduces pulmonary metastases in murine osteosarcoma. Clin Orthop Relat Res,2008,466(6):1285-91
[8]Zhang L, Gao LF, Zhao LJ, et al. Intratumoral delivery and suppression of prostate tumor growth by attenuate Sdalmonella enterica serovar typhimuriumc arrying plasmid-based small interfering RNAs. Cancer Res,2007, 67(12):5859-64
[9]King I, Bermudes D, Lin S, et al. Tumor-targeted Salmonella expressing cytosine deaminase as an anticancer agent. HumGene Ther,2002,13(10):1225-33
[10]Seger R, Krebs EG.The MAPK signaling caseade. FASEB J,1995,9(9):726-735
[11]Alejandro EU, Johnson JD. Inhibition of Raf-1 alters multiple downstream pathways to induce pancreatic beta-cell apoptosis [J] J BiolChem,2008,283(4):2407-2417.
[12]Howie HL, Shiflett SL, So M. ExtracelljiLar signal-reguLated kmase activation by Neisseria gonorrhoeae downregulates epithelial cell proapoptotic proteins Bad and Bim [J].Infect Immun,2008,76(6):2715-2721.
1 Nauts, HC, Swift, WE, and Coley, BL. The treatment of malignant tumours by bacterial toxins as developed by the late William B. Coley, MD. Cancer Res,1946, 38:9.
2 Eisentein TK, Bushnell B, Meissler JJ. Immunotherapy of a plasmacytoma with attenuated Salmonella. Med Oncol,1995,12(2):103-108.
3 Pawelek, J, Low, KB, and Bermudes. Tumor-targeted Salmonella as a novel anti-cancer vector. Cancer Res,1997,57:4537.
4 Kochi SK, Killeen KP, Pyan US, et al. Advances in the development of bacterial vector technology. Expert Rev Vaccine,2003,2(1):31-43.
5 Bermudes D, Zheng LM, King IC, Live bacteria as anticancer agents and tumor-selective protein delivery vectors. Curr Opion Drug Discov Devel,2002,5(2): 194-9.
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