[Inhibitory effect of endoplasmic reticulum-retained anti-type IV collagenase intrabody on invasion and proliferation of cancer cell].
OBJECTIVE
Invasion and metastasis are significant characteristics of cancer cells, Type IV collagenase (matrix metalloproteinase-2, and-9) plays an important role in cancer invasion and metastasis. This study was designed to block the secretion of type IV collagenase via intracellular antibody (intrabody) methods, and inhibit cancer invasion and metastasis.


METHODS
We constructed expression plasmid pcDNA3.1-ER.scFv coding for an endoplasmic reticulum (ER)- retained, single chain antibody (ER.scFv) against the type IV collagenase. The intrabody gene was transfected into human giant cell pulmonary carcinoma PG cells. Western blot was performed to detect the expression of pcDNA3.1-ER.scFv. Co-immunoprecipitation was used to analyse the interaction between ER. scFv and target protein in PG cells. The secretion of type IVcollagenase was detected by gelatin zymography. Cell behavior was examined by invasion and proliferation assay in vitro.


RESULTS
ER. scFv expressed in PG cells, and can recognize and combine its cognate target protein matrix metalloproteinase-9. Intrabody gene transfection significantly blocked the function and activity of type IV collegenase. The ER.scFv-transfected PG cells were less invasive than parental or vector control cells, the suppression rate of ER.scFv-transfected PG cells was 76.3%(P< 0.05). ER. scFv showed anti-proliferative effect on PG cells cultured on Matrigel.


CONCLUSIONS
ER-retained intrabody technology may restrain the activity of type IV collagenase in protein processing and secretory pathway, and inhibits cancer cell invasion and proliferation. Anti-type IV collagenase intrabody may be further used in cancer gene therapy.
Shen
En-Yun
EY
Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100050, PR China.
Wang
Wei-Gang
WG
Li
Yi
Y
Zhang
Sheng-Hua
SH
Zhen
Yong-Su
YS
D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis* / Q000235:genetics; D008175:Lung Neoplasms / Q000378:metabolism / Q000473:pathology*; D020778:Matrix Metalloproteinase 2 / Q000276:immunology* / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000276:immunology* / Q000378:metabolism; D014162:Transfection
D007135:Immunoglobulin Variable Region; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9