重组人Canstatin蛋白及溶瘤腺病毒He-Cans治疗胰腺癌的实验研究
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摘要
本课题旨在将抗血管生成治疗与基因治疗相结合,探讨胰腺癌治疗新策略。首先利用逆转录多聚酶链式反应(RT-PCR)从人胎盘组织扩增出新的血管生成抑制剂基因canstatin,插入质粒pET-22b(+)构建原核表达载体pET/canstatin,转化大肠杆菌,在IPTG诱导下表达出重组人Canstatin蛋白,体内实验证实其能通过抑制血管生成有效抑制胰腺癌生长。继而,将canstatin基因插入载体pCA13和pXC7C,构建出穿梭载体pCA13-Cans和pXC7C-Cans,再分别与pBGHE3共转染293细胞,获得重组非增殖型腺病毒Ad5-Cans和增殖型腺病毒(溶瘤病毒)He-Cans。体内外实验证实,溶瘤病毒He-Cans能有效抑制胰腺癌生长,疗效强于Ad5-Cans和Canstatin蛋白,且副作用少,具有良好临床应用前景。其作用机制主要是:①表达Canstatin蛋白,抑制肿瘤新生血管形成;②在肿瘤细胞中特异性增殖并裂解肿瘤细胞,从而发挥溶瘤作用。
The purpose of the present study is to develop new treatment strategy for pancreatic cancer through combining anti-angiogenesis therapy with gene therapy. The canstatin gene cDNA was amplified by RT-PCR from the total RNA extracted from human placenta, and was inserted into plasmid pET-22b(+) to construct prokaryotic expression vector pET/canstatin, which was induced to express Canstatin protein by IPTG after transformed into E. coli BL21. Our findings demonstrate that Canstatin protein effectively retards the growth of pancreatic cancer through inhibiting angiogenesis in vivo. Then, canstatin gene was inserted into vector pCA13 and pXC7C to construct shutter vector pCA13-Cans and pXC7C-Cans, which were co-transfected with pBHGE3 separately into 293 cells to produce new recombinant replication incompetent and competent adenoviruses, named as Ad5-Cans and He-Cans respectively. The results of the in vitro and in vivo experiments show that adenovirus He-Cans efficiently inhibits the growth of pancreatic cancer, far more potent than adenovirus Ad5-Cans and Canstatin protein without remarkable adverse effects, showing a promising future in its clinical application. The main mechanisms of its anti-tumor effect include anti-angiogenesis effect by expressing Canstatin protein and oncolytic effect by replicating selectively in tumor cells.
The purpose of the present study is to develop new treatment strategy for pancreatic cancer through combining anti-angiogenesis therapy with gene therapy. The canstatin gene cDNA was amplified by RT-PCR from the total RNA extracted from human placenta, and was inserted into plasmid pET-22b(+) to construct prokaryotic expression vector pET/canstatin, which was induced to express Canstatin protein by IPTG after transformed into E. coli BL21. Our findings demonstrate that Canstatin protein effectively retards the growth of pancreatic cancer through inhibiting angiogenesis in vivo. Then, canstatin gene was inserted into vector pCA13 and pXC7C to construct shutter vector pCA13-Cans and pXC7C-Cans, which were co-transfected with pBHGE3 separately into 293 cells to produce new recombinant replication incompetent and competent adenoviruses, named as Ad5-Cans and He-Cans respectively. The results of the in vitro and in vivo experiments show that adenovirus He-Cans efficiently inhibits the growth of pancreatic cancer, far more potent than adenovirus Ad5-Cans and Canstatin protein without remarkable adverse effects, showing a promising future in its clinical application. The main mechanisms of its anti-tumor effect include anti-angiogenesis effect by expressing Canstatin protein and oncolytic effect by replicating selectively in tumor cells.
引文
1. Takhar AS, Palaniappan P, Dhingsa R, et al. Recent developments in diagnosis of pancreatic cancer. BMJ. 2004, 329:668-673
2. de Braud F, Cascinu S, Gatta G. Cancer of pancreas. Crit Rev Oncol Hematol. 2004, 50: 147-155
3. MacKenzie MJ. Molecular therapy in pancreatic adenocarcinoma. Lancet Oncol. 2004, 5:541-549
4. Ranieri G, Gasparini G. Angiogenesis and angiogenesis inhibitors: a new potential anticancer therapeutic strategy. Curr Drug Targets Immune Endocr Metabol Disord 2001, 1: 241-253
5. Dudek AZ, Pawlak WZ, Kirstein MN. Molecular targets in the inhibition of angiogenesis. Expert Opin Ther Targets 2003, 7:527-541
6. Pino SM, Xiong HQ, McConkey D, Abbruzzese JL. Novel therapies for pancreatic adenocarcinoma. Curr Gastroenterol Rep. 2004, 6: 119-125
7. Onizuka S, Kawakami S, Taniguchi K, Fujioka H, Miyashita K. Pancreatic carcinogenesis: apoptosis and angiogenesis. Pancreas. 2004, 28:317-319
8. Carmeliet P. Angiogenesis in life, disease and medicine. Nature. 2005, 438:932-936
9. Soft GA. Angiostatin and angiostatin-related proteins. Cancer Metastasis Rev. 2000, 19:97-107
10. Kuwahara K, Sasaki T, Kuwada Y, Murakami M, Yamasaki S, Chayama K.Expressions of angiogenic factors in pancreatic ductal carcinoma: a correlative study with clinicopathologic parameters and patient survival. Pancreas. 2003, 26: 344-349
11. Cao Y, Cao R, Veitonmaki N. Kringle structures and antiangiogenesis. Curr Med Chem Anti-Canc Agents. 2002, 2: 667-681
12. Sim BKL, MacDonald NJ, Gubish ER. Angiostatin and Endostatin: Endogenous inhibitors of tumor growth. Cancer Metastasis Rev 2000, 19:181 190
13. Kirsch M, Schackert G, Black PM. Angiogenesis, metastasis, and endogenous inhibition. J Neurooncol 2000, 50: 173-180
14. Ren B, Hoti N, Rabasseda X, Wang YZ, Wu M. The antiangiogenic and therapeutic implications of endostatin. Methods Find Exp Clin Pharmacol 2003, 25: 215-224
15. Hansma AH, Broxterman HJ, van der Horst I, Yuana Y, Boven E, Giaccone G, Pinedo HM, Hoekman K. Recombinant human endostatin administered as a 28-day continuous intravenous infusion, followed by daily subcutaneous injections: a phase I and pharmacokinetic study in patients with advanced cancer. Ann Oncol. 2005, 16:1695-1701.
16. Tsuchida Y, Shitara T, Kuroiwa M, Ikeda H. Current treatment and future directions in neuroblastoma. Indian J Pediatr. 2003, 70:809-812.
17. Thomas JP, Arzoomanian RZ, Alberti D, Marnocha R, Lee F, Friedl A, Tutsch K, Dresen A, Geiger P, Pluda J, Fogler W, Schiller JH, Wilding G. Phase I pharmacokinetic and pharmacodynamic study of recombinant human endostatin in patients with advanced solid tumors. J Clin Oncol. 2003, 21(2):223-231
18. Herbst RS, Mullani NA, Davis DW, Hess KR, McConkey D J, Charnsangavej C, O'Reilly MS, Kim HW, Baker C, Roach J, Ellis LM, Rashid A, Pluda J, Bucana C, Madden TL, Tran HT, Abbruzzese JL. Development of biologic markers of response and assessment of antiangiogenic activity in a clinical trial of human recombinant endostatin. J Clin Oncol. 2002, 20:3804-3814
19. Kamphaus GD, Colorado PC, Panka DJ, et al. Canstatin, a novel matrix-derived inhibitor of angiogenesis and tumor growth. J Biol Chem. 2000, 275:1209-1215
20. Kong HL, Crystal RG. Gene therapy strategies for tumor antiangiogenesis. J Natl Cancer Inst. 1998, 90:273-286
21. Nesbit M. Abrogation of tumor vasculature using gene therapy. Cancer Metastasis Rev. 2000,19: 45-49
22. Yoon AR, Kim JH, Lee YS, Kim H, Yoo JY, Sohn JH, Park BW, Yun CO. Markedly Enhanced Cytolysis by E1B-19kD-Deleted Oncolytic Adenovirus in Combination with Cisplatin. Hum Gene Ther. 2006, 17:379-390.
23. Glockzin G, Mantwill K, Jurchott K, Bernshausen A, Ladhoff A, Royer HD, Gansbacher B, Holm PS. Characterization of the recombinant adenovirus vector AdYB-1: implications for oncolytic vector development. J Virol. 2006, 80: 3904-3911.
2. de Braud F, Cascinu S, Gatta G. Cancer of pancreas. Crit Rev Oncol Hematol. 2004, 50: 147-155
3. MacKenzie MJ. Molecular therapy in pancreatic adenocarcinoma. Lancet Oncol. 2004, 5:541-549
4. Ranieri G, Gasparini G. Angiogenesis and angiogenesis inhibitors: a new potential anticancer therapeutic strategy. Curr Drug Targets Immune Endocr Metabol Disord 2001, 1: 241-253
5. Dudek AZ, Pawlak WZ, Kirstein MN. Molecular targets in the inhibition of angiogenesis. Expert Opin Ther Targets 2003, 7:527-541
6. Pino SM, Xiong HQ, McConkey D, Abbruzzese JL. Novel therapies for pancreatic adenocarcinoma. Curr Gastroenterol Rep. 2004, 6: 119-125
7. Onizuka S, Kawakami S, Taniguchi K, Fujioka H, Miyashita K. Pancreatic carcinogenesis: apoptosis and angiogenesis. Pancreas. 2004, 28:317-319
8. Carmeliet P. Angiogenesis in life, disease and medicine. Nature. 2005, 438:932-936
9. Soft GA. Angiostatin and angiostatin-related proteins. Cancer Metastasis Rev. 2000, 19:97-107
10. Kuwahara K, Sasaki T, Kuwada Y, Murakami M, Yamasaki S, Chayama K.Expressions of angiogenic factors in pancreatic ductal carcinoma: a correlative study with clinicopathologic parameters and patient survival. Pancreas. 2003, 26: 344-349
11. Cao Y, Cao R, Veitonmaki N. Kringle structures and antiangiogenesis. Curr Med Chem Anti-Canc Agents. 2002, 2: 667-681
12. Sim BKL, MacDonald NJ, Gubish ER. Angiostatin and Endostatin: Endogenous inhibitors of tumor growth. Cancer Metastasis Rev 2000, 19:181 190
13. Kirsch M, Schackert G, Black PM. Angiogenesis, metastasis, and endogenous inhibition. J Neurooncol 2000, 50: 173-180
14. Ren B, Hoti N, Rabasseda X, Wang YZ, Wu M. The antiangiogenic and therapeutic implications of endostatin. Methods Find Exp Clin Pharmacol 2003, 25: 215-224
15. Hansma AH, Broxterman HJ, van der Horst I, Yuana Y, Boven E, Giaccone G, Pinedo HM, Hoekman K. Recombinant human endostatin administered as a 28-day continuous intravenous infusion, followed by daily subcutaneous injections: a phase I and pharmacokinetic study in patients with advanced cancer. Ann Oncol. 2005, 16:1695-1701.
16. Tsuchida Y, Shitara T, Kuroiwa M, Ikeda H. Current treatment and future directions in neuroblastoma. Indian J Pediatr. 2003, 70:809-812.
17. Thomas JP, Arzoomanian RZ, Alberti D, Marnocha R, Lee F, Friedl A, Tutsch K, Dresen A, Geiger P, Pluda J, Fogler W, Schiller JH, Wilding G. Phase I pharmacokinetic and pharmacodynamic study of recombinant human endostatin in patients with advanced solid tumors. J Clin Oncol. 2003, 21(2):223-231
18. Herbst RS, Mullani NA, Davis DW, Hess KR, McConkey D J, Charnsangavej C, O'Reilly MS, Kim HW, Baker C, Roach J, Ellis LM, Rashid A, Pluda J, Bucana C, Madden TL, Tran HT, Abbruzzese JL. Development of biologic markers of response and assessment of antiangiogenic activity in a clinical trial of human recombinant endostatin. J Clin Oncol. 2002, 20:3804-3814
19. Kamphaus GD, Colorado PC, Panka DJ, et al. Canstatin, a novel matrix-derived inhibitor of angiogenesis and tumor growth. J Biol Chem. 2000, 275:1209-1215
20. Kong HL, Crystal RG. Gene therapy strategies for tumor antiangiogenesis. J Natl Cancer Inst. 1998, 90:273-286
21. Nesbit M. Abrogation of tumor vasculature using gene therapy. Cancer Metastasis Rev. 2000,19: 45-49
22. Yoon AR, Kim JH, Lee YS, Kim H, Yoo JY, Sohn JH, Park BW, Yun CO. Markedly Enhanced Cytolysis by E1B-19kD-Deleted Oncolytic Adenovirus in Combination with Cisplatin. Hum Gene Ther. 2006, 17:379-390.
23. Glockzin G, Mantwill K, Jurchott K, Bernshausen A, Ladhoff A, Royer HD, Gansbacher B, Holm PS. Characterization of the recombinant adenovirus vector AdYB-1: implications for oncolytic vector development. J Virol. 2006, 80: 3904-3911.