ABSTRACT
Tissue Factor Pathway Inhibitor-2 (TFPI-2) is a potent inhibitor of plasmin which activates metalloproteinases (MMPs) involved in extracellular matrix (ECM) degradation. Its secretion in ECM makes TFPI-2 a potential inhibitor to regulate tumour invasion and metastasis. Moreover, TFPI-2 is frequently downregulated, particularly in aggressive cancers. In this study, we silenced TFPI-2 in the NCI-H460 non-small cell lung cancer cell line and evaluated the role of TFPI-2 in cell invasion and its impact on MMPs expression. As the effects of siRNA are transient, the consequences of both gene silencing and restoration to normal expression could be studied kinetically in the same cells. We showed that TFPI-2 expression by NCI-H460 cells was effectively downregulated using specific small interfering RNA and this silencing was associated with an increase in the invasive potential of tumour cells while migration was not affected. We also showed that mRNA levels and protein expression of MMP-2, -3, -9, -14 were not influenced by TFPI-2 silencing. Moreover, the gelatinase activity of MMP-2 and MMP-9 was unmodified. In contrast, MMP-1 mRNA levels and protein were significantly and similarly increased in cells transfected with TFPI-2 siRNA. In conclusion, this study confirms that TFPI-2 downregulation can contribute to tumour invasion of lung cancer cells.
Subject(s)
Carcinoma, Non-Small-Cell Lung/metabolism , Gene Expression Regulation, Neoplastic , Gene Silencing , Glycoproteins/metabolism , Lung Neoplasms/metabolism , RNA/metabolism , Carcinoma, Non-Small-Cell Lung/pathology , Cell Line, Tumor , Cell Movement , Down-Regulation , Extracellular Matrix/metabolism , Humans , Lung Neoplasms/pathology , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinase 9/metabolism , Neoplasm Invasiveness , RNA, Small Interfering/metabolismABSTRACT
A range of gene delivery vectors containing the thermoresponsive polymer, poly(N-isopropylacrylamide) (PNIPAm) was evaluated for effects on cell viability, intracellular trafficking and transgene expression in C2C12 mouse muscle cells. Polymers were complexed with plasmid DNA at pH 7.4 and the ability of the resulting particles to transfect cells was assessed via confocal microscopy and protein expression studies in tissue culture. Cell viability assays indicated that these polymers were toxic at high concentrations when not complexed to DNA or at certain polymer:DNA ratios. Poly(ethyleneimine) co-polymers with side-chain grafted PNIPAm were shown to be less toxic than poly(ethyleneimine) alone or PNIPAm-co-(N,N'-dimethylaminoethylmethacrylate) linear co-polymers and the effects were concentration dependent. Confocal micrographs of labeled polymers and DNA indicated rapid cellular entry for all the complexes but expression of Green Fluorescent Protein was achieved only when the branched PEI-PNIPAm co-polymers were used as vectors. The results indicate that design of appropriate co-polymer components and overall polymer architecture can be used to mediate, and perhaps ultimately control, DNA transport and transgene expression.
