目的 观察抗明胶酶dFv-LDP以及其强化融合蛋白dFv-LDP-AE对人纤维肉瘤HT-1080的生长抑制作用。方法 采用蛋白质印迹法分析不同细胞系中明胶酶表达情况,四甲基偶氮唑蓝法分析融合蛋白dFv-LDP以及强化融合蛋白dFv-LDP-AE的对HT-1080的生长抑制作用;酶联免疫吸附法以及免疫荧光分析融合蛋白dFv-LDP和HT-1080的结合能力;流式细胞术分析两者单独或者联合对HT-1080的周期阻滞;评价融合蛋白dFv-LDP和dFv-LDP-AE单独和联合体内抗HT-1080Luc肺转移的活性。结果 HT-1080的明胶酶表达丰度较之其他类型肿瘤明显,酶联免疫吸附法和免疫荧光分析表明融合蛋白dFv-LDP与HT-1080细胞有着良好的亲和力;四甲基偶氮唑蓝法结果显示dFv-LDP-AE对HT-1080体外增殖有着强烈的杀伤作用;FACS表明融合蛋白dFv-LDP和dFv-LDP-AE的联合对细胞周期的阻滞没有明显的增效作用;体内实验性肺转移实验结果表明,融合蛋白dFv-LDP(10 mg·kg-1)组肺部转移灶数目为对照组的55.8%(P<0.01);强化融合蛋白dFv-LDP-AE在0.4和0.6 mg·kg-1剂量下的转移灶数目分别为对照组的41.4%(与dFv-LDP 10 mg·kg-1组相比,P<0.05)和25.1%(与dFv-LDP 10 mg·kg-1组相比,P<0.05);联合dFv-LDP后,其肺部转移灶数目为对照组的20.3%(与dFv-LDP-AE 0.4 mg·kg-1组相比,P<0.05)和13.1% (与dFv-LDP-AE 0.6 mg·kg-1组相比,P<0.05),提示联合给药可进一步提高抗转移的效果。结论 融合蛋白dFv-LDP联合强化融合蛋白dFv-LDP-AE对裸鼠移植HT-1080实验性肺转移的抑制效果具有增效作用。
Abstract
OBJECTIVE To investigate the antitumor efficacy of anti-gelatinases dFv-LDP and its enediyne-energized fusion protein dFv-LDP-AE on human fibrosarcoma HT-1080 cancer cells. METHODS Western blot was used to analyze the expression level of gelatinases in different cancer cell lines. The inhibitory effects of fusion protein dFv-LDP and its enediyne-energized fusion protein dFv-LDP-AE on HT-1080 were determined by MTT assay. The binding capability of fusion protein dFv-LDP with HT-1080 was detected by ELISA and immunofluorescence. FACS was used to analyze the cell cycle arrest by dFv-LDP, dFv-LDP-AE or their combination on HT-1080 cells. The anti-metastasis effects of dFv-LDP, dFv-LDP-AE or their combination on the experimental lung metastasis model established by HT-1080Luc via tail vein injection were also evaluated in this study. RESULTS Expression level of gelatinases was higher in HT-1080 cells as compared to that of other cancer cell lines. The fusion protein dFv-LDP showed well binding capability with HT-1080 cells as determined by ELISA and immunofluorescence. The enediyne-energized fusion protein dFv-LDP-AE displayed extremely inhibitory effect on proliferation of HT-1080. Results of FACS indicated that the combination of dFv-LDP with dFv-LDP-AE could not further increase the G2/M proportion on cell cycle arrest. However, in vivo experiment as examined using the experimental lung metastasis model established via HT-1080Luc tail veil injection, the metastasis foci in group of fusion protein dFv-LDP (10 mg穔g-1) was 55.8% compared to that of control group (P<0.01). The metastasis foci in group of dFv-LDP-AE at dosage of 0.4 and 0.6 mg穔g-1 were 41.4% and 25.1% respectively compared to that of dFv-LDP 10 mg穔g-1 group (P<0.01). The combination of dFv-LDP (10 mg穔g-1) with dFv-LDP-AE (0.4 or 0.6 mg穔g-1) showed an additive decrease of metastasis foci number in the lung of athymic mice, which were 20.3% (P<0.05,compared with dFv-LDP-AE at 0.4 mg穔g-1) and 13.1% (P<0.05,compared with dFv-LDP-AE at 0.6 mg穔g-1) respectively. CONCLUSION The combination of dFv-LDP with its enediyne-energized fusion protein dFv-LDP-AE would intensify the anti-metastasis effect on experimental lung metastasis model as established via tail vein injection of HT-1080Luc cells.
关键词
力达霉素 /
明胶酶 /
纤维肉瘤 /
实验性肺转移
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Key words
lidamycin /
geltainases /
fibrosarcoma /
experimental lung metastasis
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中图分类号：
R965
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参考文献
[1] DERYUGINA E I, QUIGLEY J P. Matrix metalloproteinases and tumor metastasis . Cancer Metastasis Rev, 2006, 25(1):9-34. [2] MATRISIAN L M. Matrix metalloproteinase gene expression . Ann N Y Acad Sci, 1994, 732:42-50. [3] AHN G O, BROWN J M. Matrix metalloproteinase-9 is required for tumor vasculogenesis but not for angiogenesis: Role of bone marrow-derived myelomonocytic cells . Cancer Cell, 2008, 13(3):193-205. [4] KESSENBROCK K, PLAKS V, WERB Z. Matrix metalloproteinases: Regulators of the tumor microenvironment . Cell, 2010, 141(1):52-67. [5] SHAO R G, ZHEN Y S. Enediyne anticancer antibiotic lidamycin: Chemistry, biology and pharmacology . Anticancer Agents Med Chem, 2008, 8(2):121-131. [6] BEERMAN T A, GAWRON L S, SHIN S, et al. C-1027, a radiomimetic enediyne anticancer drug, preferentially targets hypoxic cells . Cancer Res, 2009, 69(2):593-598. [7] GUO X F, ZHU X F, SHANG Y, et al. A bispecific enediyne-energized fusion protein containing ligand-based and antibody-based oligopeptides against epidermal growth factor receptor and human epidermal growth factor receptor 2 shows potent antitumor activity . Clin Cancer Res, 2010, 16(7):285-2094. [8] ZHONG G, ZHANG S, LI Y, et al. A tandem scFv-based fusion protein and its enediyne-energized analogue show intensified therapeutic efficacy against lung carcinoma xenograft in athymic mice . Cancer Lett, 2010, 295(1):124-133. [9] GAO R J, LI L, ZHAO C Y, et al. Antitumor effect of the engineered and energized fusion protein Fv-LDP-AE composed of lidamycin and anti-type IV collagenase single-chain Fv antibody . Chin Pharm J (中国药学杂志), 2010, 45(11):808-813. WU A M, OLAFSEN T. Antibodies for molecular imaging of cancer . Cancer J, 2008, 14(3):191-197. LI L, YAZAKI P J, ANDERSON A L, et al. Improved biodistribution and radioimmunoimaging with poly(ethyleneglycol)-DOTA-conjugated anti-CEA diabody . Bioconjugate Chem, 2006, 17(1):68-76. ZHONG G, GUO X, ZHANG S, et al. Optimization of the assembly efficiency for lidamycin chromophore bound to its apoprotein: A case study using orthogonal array . Biomed Environ Sci, 2011, 24(6):408-414. THORNTON K. Chemotherapeutic management of soft tissue sarcoma . Surg Clin North Am, 2008, 88(3):647-660. MAKI R G. Role of chemotherapy in patients with soft tissue sarcomas . Expert Rev Anticancer Ther, 2004, 4(2):229-236. ZHANG S H, ZHONG G S, HE H W, et al. Bioluminescence imaging evaluation of the inhibitory effect of lidamycin on lung metastasis of human fibrosacroma in athymic mice . Acta Pharm Sin (药学学报), 2011, 46(1):45-49. COLE P D, KAMEN B A, GORLICK R, et al. Effects of overexpression of gamma-Glutamyl hydrolase on methotrexate metabolism and resistance . Cancer Res, 2001, 61(1):4599-4604. COLE P D, SMITH A K, KAMEN B A. Osteosarcoma cells, resistant to methotrexate due to nucleoside and nucleobase salvage, are sensitive to nucleoside analogs . Cancer Chemother Pharmacol, 2002, 50(2):111-116. BERTINO J R, GOKER E, GORLICK R, et al. Resistance mechanisms to methotrexate in tumors . Stem Cells, 1996, 14(1):5-9. CORREIA A L, BISSELL M J. The tumor microenvironment is a dominant force in multidrug resistance . Drug Resist Updat, 2012, 15(1-2):39-49. MEADS M B, GATENBY R A, DALTON W S. Environment-mediated drug resistance: A major contributor to minimal residual disease . Nat Rev Cancer, 2009, 9(9):665-674.
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脚注
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基金
国家科技重大专项“重大新药创制”(2009ZX09301-003;2009ZX09401-005);国家自然科学青年基金(81201765)
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