[Inhibitory effect of anti-type IV collagenase intrabody on invasiveness of human pulmonary giant cell carcinoma PG cells in vitro].
OBJECTIVE
To explore the inhibitory effects of endoplasmic reticulum-retained intrabody on the secretion of type IV collagenase and the invasion of human pulmonary giant cell carcinoma PG cells in vitro.


METHODS
Two expression plasmids were constructed, pcDNA3.1-CP.scFv and pcDNA3.1-ER.scFv encoding cytoplasm-retained and endoplasmic reticulum-retained single chain antibodies against the type IV collagenase, respectively. The intracellular antibody genes were transfected into the human pulmonary giant cell carcinoma PG cells. Western blot was performed to detect the expression of pcDNA3.1-CP.scFv and pcDNA3.1-ER.scFv. Gelatin zymography was performed to detect seretion of type IV collagenase in PG cells and Matrigel assay was employed for determination of the cell invasiveness.


RESULTS
Both of cytoplasm-retained and endoplasmic reticulum-retained introbodies, CP.scFv and ER.scFv, were expressed in PG cells. ER.scFv, significantly inhibited the secretion of type IV collegenase. As shown, matrix metalloproteinase 9 and matrix metalloproteinase 2 were inhibited by 85.7% and by 51.2%, respectively. However, CP.scFv did not show such inhibitory effect. The ER.scFv encoding gene-transfected PG cells were much less invasive than parental or vector control cells, the inhibition rate was 76.3% (P < 0.05), whereas CP.scFv encoding gene-transfected PG cells showed no reduction in invasiveness.


CONCLUSIONS
Those findings demonstrate that endoplasmic reticulum (ER)-retained intracellular antibody technology may selectively abrogate the activity of type IV collagenase in the protein trafficking and secretory pathway and effectively inhibit tumor cell invasion in vitro. 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 and Peking Union Medical College, Beijing 100050, China.
Wang
Wei-gang
WG
Zhang
Sheng-hua
SH
Zhen
Yong-su
YS
D018286:Carcinoma, Giant Cell / Q000378:metabolism / Q000473:pathology*; D045744:Cell Line, Tumor; D003593:Cytoplasm / Q000276:immunology; D004721:Endoplasmic Reticulum / Q000276:immunology; D005822:Genetic Vectors; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000378:metabolism* / Q000502:physiology; D008175:Lung Neoplasms / Q000378:metabolism / Q000473:pathology*; D020778:Matrix Metalloproteinase 2 / Q000276:immunology / Q000378:metabolism*; D020780:Matrix Metalloproteinase 9 / Q000276:immunology / Q000378:metabolism*; D009361:Neoplasm Invasiveness; D010957:Plasmids; D014162:Transfection
D007135:Immunoglobulin Variable Region; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9