Although mounting evidence indicates the involvement of galectin-3 in cancer progression and metastasis, the underlying molecular mechanisms remain largely unknown. In this study, we investigated the effect and possible mechanism of galectin-3 on the migration and invasion of B16F10, a metastatic melanoma cell line, in which galectin-3 and matrix metalloproteinase-1 (MMP-1) were both found to be highly expressed. Knockdown of galectin-3 with specific siRNA reduced migration and invasion, which was associated with reduced expression of MMP-1. To further investigate the underlying mechanism, we examined the effect of galectin-3 knockdown on the activity of AP-1, a transcriptional factor regulating MMP-1 expression. We found that galectin-3 directly interacted with AP-1 and facilitated the binding of this complex to the MMP-1 promoter that drives MMP-1 transcription. Moreover, silencing of galectin-3 inhibited binding of fra-1 and c-Jun to promoter sites of MMP-1 gene. Consistent with these in vitro findings, our in vivo study demonstrated that galectin-3 shRNA treatment significantly reduced the total number of mouse lung metastatic nodules. Taken together, galectin-3 facilitates cell migration and invasion in melanoma in vitro and can induce metastasis in vivo, in part through, regulating the transcription activity of AP-1 and thereby up-regulating MMP-1 expression.
Animals ; Binding Sites/genetics ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Galectin 3/genetics/*metabolism ; JNK Mitogen-Activated Protein Kinases/metabolism ; Lung Neoplasms/drug therapy/genetics/*secondary ; Matrix Metalloproteinase 1/*genetics/*metabolism ; Melanoma, Experimental/*metabolism/*pathology/secondary ; Mice ; Mice, Inbred C57BL ; NIH 3T3 Cells ; Neoplasm Metastasis ; Promoter Regions, Genetic ; Proto-Oncogene Proteins c-fos/metabolism ; RNA Interference ; RNA, Small Interfering ; Transcription Factor AP-1/*genetics/metabolism ; Transcription, Genetic ; Transcriptional Activation

OBJECTIVETo investigate the inhibitory effect of DNA vaccine immunization on neu-overexpressed melanoma growth in prophylactic treatment and anti-lung-metastasis experiments in C57BL/6 mice.

METHODSpcDNA-neu transfected into B16F10 with transfection reagent Fugene 6, neu-overexpressed cell clone B16F10-neu was selected with limited dilution method. The growth curve was drawn to analyse its proliferating character in vitro. With Helios gene gun system, DNA vaccine pWRG-neu was immunized to 8-week-old C57BL/6 mice in the shaved abdominal skin for 3 times at two-weekly interval. After immunization, the life span was analyzed. Using MTT assay, the cytolysis activity of the DNA immunized mice spleen cells was compared.

RESULTSOne clone of neu-overexpressed B16F10-neu was selected and its proliferating character was the same as B16F10 and B16F10-pcDNA. In prophylactic, treatment and anti-lung-metastasis experiments, gene gun-mediated pWRG-neu immunization could exhibit antitumor effects. The growth and metastasis of neu-overexpressed melanoma was reduced dramatically. The spleen cells of the immuned mice showed cytotoxic T lymphocyte (CTL) activity.

CONCLUSIONGene gun-mediated gene transfer is effective and practicable. DNA vaccine pWRG-neu is potent in preventing subsequent tumor cells challenge, inhibiting the tumor growth and metastasis.

Animals ; Biolistics ; Cell Line, Tumor ; Cytotoxicity, Immunologic ; Genes, erbB-2 ; Immunization ; Lung Neoplasms ; prevention & control ; secondary ; Melanoma, Experimental ; metabolism ; pathology ; therapy ; Mice ; Mice, Inbred C57BL ; Neoplasm Transplantation ; Plasmids ; Receptor, ErbB-2 ; metabolism ; T-Lymphocytes, Cytotoxic ; immunology ; Vaccines, DNA