title: Effects of recombinant human canstatin protein in the treatment of pancreatic cancer.
abstract: OBJECTIVE
  To examine the effect of canstatin, a newly discovered endogenous inhibitor of angiogenesis, in the treatment of pancreatic cancer in vivo.
  
  
  METHODS
  The canstatin cDNA fragment was synthesized and amplified from the total RNA extracted from human placenta tissues by RT-PCR. The resulting product was firstly cloned into pUCm-T vector, then into plasmid pET-22b (+) and transformed into E. coli BL21. Isopropyl-1-thio-b-Dgalactopyran-oside (IPTG) was used to induce the expression of canstatin protein and affinity chromatography was used to purify the protein. To determine the activity of purified recombinant human canstatin (rhCanstatin), orthotopic xenograft human pancreatic cancer models were established. Human pancreatic cancer cells (SW1990) were injected into the pancreas of BALB/c nude mice. Twenty-four nude mice with orthotopic xenograft tumor were randomly divided into 3 groups 10 d after the inoculation, and were treated with PBS 0.3 mL, or canstatin 5 mg/kg, or 10 mg/kg per day for 3 wk intraperitoneally. When the experiment was over, all tumors were resected and the effects of rhCanstatin on tumor growth, microvessel density (MVD) were analyzed.
  
  
  RESULTS
  After IPTG induction, SDS-PAGE showed a new monomeric 24 kDa protein band. This protein was purified through affinity chromatography and refolded through dialysis with a final concentration of 60 mg/L. In orthotopic pancreatic cancer models, the final tumor volume in groups treated with PBS, canstatin 5 mg/kg, 10 mg/kg were 355.21+/-39.54 mm3, 112.73+/-10.47 mm3, and 61.75+/-6.99 mm3 respectively. The immunohistochemical examination showed that the MVD in tumors treated with canstatin was significantly less than that in other group.
  
  
  CONCLUSIONS
  These findings demonstrate that the rhCanstatin effectively retards the growth of pancreatic cancer in a dose-dependent manner through inhibiting angiogenesis and may be a promising therapeutic agent for pancreatic cancer treatment in the clinic.
authors.affiliation: Department of Gastroenterology, Nanjing General Hospital, Jiangsu Province, and Second Military Medical University, Shanghai, China.
authors.forename: Xiao-Ping
authors.initials: XP
authors.lastname: He
authors.forename: Zhao-Shen
authors.initials: ZS
authors.lastname: Li
authors.forename: Ren-Min
authors.initials: RM
authors.lastname: Zhu
authors.forename: Zhen-Xing
authors.initials: ZX
authors.lastname: Tu
authors.forename: Jun
authors.initials: J
authors.lastname: Gao
authors.forename: Xue
authors.initials: X
authors.lastname: Pan
authors.forename: Yan-Fang
authors.initials: YF
authors.lastname: Gong
authors.forename: Jing
authors.initials: J
authors.lastname: Jin
authors.forename: Xiao-Hua
authors.initials: XH
authors.lastname: Man
authors.forename: Hong-Yu
authors.initials: HY
authors.lastname: Wu
authors.forename: Ai-Fang
authors.initials: AF
authors.lastname: Xu
mesh_terms: D045744:Cell Line, Tumor; D049109:Cell Proliferation; D024141:Collagen Type IV / Q000235:genetics / Q000627:therapeutic use*; D018076:DNA, Complementary / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D009389:Neovascularization, Pathologic / Q000188:drug therapy; D010190:Pancreatic Neoplasms / Q000188:drug therapy* / Q000473:pathology; D010446:Peptide Fragments / Q000235:genetics / Q000627:therapeutic use*; D010957:Plasmids / Q000235:genetics; D012313:RNA / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000627:therapeutic use*; D023041:Xenograft Model Antitumor Assays
chemical_list: C502625:COL4A2 protein, human; D024141:Collagen Type IV; D018076:DNA, Complementary; D010446:Peptide Fragments; D011994:Recombinant Proteins; D012313:RNA