OBJECTIVETo investigate the effect of membrane type-1 matrix metalloproteinase (MTI-MMP) on the invasive potential of breast cancer cell and analyze its mechanisms.

METHODSAfter treatment of breast cancer MDA-MB-453 cell line with concanavalin A ( ConA, 20 microg/ml) for 24 h, MT1-MMP protein was detected in cancer cells by Western analysis and immunocytochemistry. MDA-MB-453 cells were cultured with exogenous latent proMMP-2 and MMP-2 activity was analyzed by gelatin zymography. The invasive potential of the tumor cells was measured with a membrane invasion culture system. Cancer cells of the cell line were divided into four groups: the control group treated by neither reagent, group ConA was only treated by ConA, group MMP-2 was treated only by MMP-2, and group ConA + MMP-2 was treated by both ConA and MMP-2. RESULTS The expression of MTI-MMP protein could be detected in groups ConA and ConA + MMP-2, but nothing was detected in control and group MMP-2. There was only 72 000 precursor form of MMP-2 in group MMP-2 and there were both 72 000 precursor form and 64 000 active enzyme form of MMP-2 in group ConA + MMP-2, but there was no forms of MMP-2 in the other two groups detected by gelatin zymography. The largest amount of cells penetrated through Matrigel was observed in group ConA + MMP-2 than in the other three groups.

CONCLUSIONMTI-MMP can remarkably promote the invasive potential of breast cancer cells mainly through its ability of activating latent proMMP-2 to degrade

Blotting, Northern ; Blotting, Western ; Breast Neoplasms ; enzymology ; genetics ; pathology ; Cell Line, Tumor ; Cell Movement ; drug effects ; Concanavalin A ; pharmacology ; Female ; Humans ; Immunohistochemistry ; Matrix Metalloproteinase 14 ; genetics ; metabolism ; Matrix Metalloproteinase 2 ; metabolism ; Neoplasm Invasiveness ; RNA, Messenger ; genetics ; metabolism

Objective:To construct the recombinant DNA E.coli ghosts (EBGs) expressing Treponema pallidum adhesin Tp0751 (pcD/Tp0751-BG) and determine its immunocompetence in immunized mice,and provide a potential novel method for syphilis vaccine developing.Methods:The recombinant eukaryotic expression plasmid pcDNA3.1(+)/Tp0751 was constructed and loaded into empty EBGs to create pcD/Tp0751-BG.The loading rate was determined accordingly.Macrophages cell line RAW264.7 was transfected with pcD/Tp0751-BG,and the expression of recombinant Tp0751(rTp0751) protein was detected by Western blot(WB).For immuno-competence in mice,the female BALB/c mice were randomly divided into 6 groups,including three control groups,A (PBS),B (EBG),C (empty pcDNA),and experimental group D(naked pcD/Tp0751),E (pcD/Tp0751-BG) and F (pcD/Tp0751-BG+rTp0751).All the mice were immunized as indicated for three times by intramuscular injection at two weeks intervals.The levels of specific IgG in sera and SIgA in genital tract lavage fluid were measured by ELISA.Levels of lymphocyte proliferation and IFN-γ secretion in spleen cells were measured by CCK-8 Cell Counting Kit and ELISA as well,respectively.Results:The loading rate of pcD/Tp0751 to EBGs was 76.1%.WB showed that the target recombinant protein pcD/Tp0751 expressed in RAW264.7 cells was active with Tp-infected rabbit sera.The titers of specific IgG and SIgA in group D,E,F gradually increased to significantly higher level as compared to the control groups (P<0. 01),which reached its peak at wk 8 after last immunization(the titers of IgG and SIgA were 1 :102 400 and 1 :12 800 in group F,respectively). Higher levels of specific IgG and SIgA were observed in groups E and F as compared to group D after first boost (P<0. 01),with groups F higher than group E after last boost(P<0. 01). At wk 8 after the last boost,the stimulation index (SI) and levels of IFN-γ in group D,E,F were all significant higher than the control groups (P<0. 01), with group E and F higher than group D (P<0. 01),and group E higher than group F (P<0. 05). Conclusion: The recombinant DNA EBGs of T. pallidum adhesin Tp0751 (pcD/ Tp0751-BG) possesses the immunocompetence to induce not only strong mucosal and systemic humoral immune response but also systemic cellular immune response in BALB/ c mice. The heterologous boost can be more efficient than homologous boost during immunization process.