Expression of angiostatin cDNA in human gallbladder carcinoma cell line GBC-SD and its effect on endothelial proliferation and growth.
OBJECTIVE
To explore the influence of angiostatin up-regulation on the biologic behavior of gallbladder carcinoma cells in vitro and in vitro, and the potential value of angiostatin gene therapy for gallbladder carcinoma.


METHODS
A eukaryotic expression vector of pcDNA3.1(+) containing murine angiostatin was constructed and identified by restriction endonuclease digestion and sequencing. The recombinant vector pcDNA3.1-angiostatin was transfected into human gallbladder carcinoma cell line GBC-SD with Lipofectamine 2000, and paralleled with the vector and mock control. The resistant clone was screened by G418 filtration. Angiostatin transcription and protein expression were examined by RT-PCR, immunofluorescence and Western-blot. The supernatant was collected to treat endothelial cells. Cell proliferation and growth in vitro were observed under microscope.


RESULTS
Murine angiostatin cDNA was successfully cloned into the eukaryotic expression vector pcDNA3.1 (+). After 14 d of transfection and selection with G418, macroscopic resistant cell cloning was formed in the experimental group transfected with pcDNA 3.1(+)-angiostatin and vector control. But untreated cells died in the mock control. Angiostatin was detected by RT-PCR and protein expression was detected in the experimental group by immunofluorescence and Western-blot. Cell proliferation and growth in vitro in the three groups were observed respectively under microscope. No significant difference was observed in the growth speed of GBC-SD cells between groups that were transfected with and without angiostatin. After treatment with supernatant, significant differences were observed in endothelial cell (ECV-304) growth in vitro. The cell proliferation and growth were inhibited.


CONCLUSIONS
Angiostatin does not directly inhibit human gallbladder carcinoma cell proliferation and growth in vitro, but the secretion of angiostatin inhabits endothelial cell proliferation and growth.
Yang
Ding-Zhong
DZ
Shannxi Provincial Hospital, Xi'an 710065, Shannxi Province, China. doctoryang8@126.com
He
Jing
J
Zhang
Ji-Cheng
JC
Wang
Zuo-Ren
ZR
D043168:Angiostatins / Q000235:genetics* / Q000502:physiology; D015153:Blotting, Western; D002277:Carcinoma / Q000737:chemistry / Q000235:genetics* / Q000473:pathology; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000032:analysis* / Q000235:genetics; D004273:DNA, Neoplasm / Q000032:analysis* / Q000235:genetics; D004730:Endothelium, Vascular / Q000166:cytology* / Q000254:growth & development; D005706:Gallbladder Neoplasms / Q000737:chemistry / Q000235:genetics* / Q000473:pathology; D015972:Gene Expression Regulation, Neoplastic; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007150:Immunohistochemistry; D008856:Microscopy, Fluorescence; D009389:Neovascularization, Pathologic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D015854:Up-Regulation
D018076:DNA, Complementary; D004273:DNA, Neoplasm; D043168:Angiostatins