Downregulation of uPA, uPAR and MMP-9 using small, interfering, hairpin RNA (siRNA) inhibits glioma cell invasion, angiogenesis and tumor growth

The diffuse, extensive infiltration of malignant gliomas into the surrounding normal brain is believed to rely on modification of the proteolysis of extracellular matrix components. Our previous results clearly demonstrate that uPA, uPAR and MMP-9 concentrations increase significantly during tumor progression and that tumor growth can be inhibited with antisense stable clones of these molecules. Because antisense-mediated gene silencing does not completely inhibit the translation of target mRNA and high concentrations of antisense molecules are required to achieve gene silencing, we used the RNAi approach to silence uPA, uPAR and MMP-9 in this study. We examined a cytomegalovirus (CMV) promoter-driven DNA-template approach to induce hairpin RNA (hpRNA)-triggered RNAi to inhibit uPA, uPAR and MMP-9 gene expression with a single construct. uPAR protein levels and enzymatic activity of uPA and MMP-9 were found to significantly decrease in cells transfected with a plasmid expressing hairpin siRNA for uPAR, uPA and MMP-9. pU(2)M-transfected SNB19 cells significantly decreased uPA, uPAR and MMP-9 expression compared to mock and EV/SV-transfected cells, determined by immunohistochemical analysis. Furthermore, the effect of the single constructs for these molecules was a specific inhibition of their respective protein levels, as demonstrated by immunohistochemical analysis. After transfection with a plasmid vector expressing dsRNA for uPA, uPAR and MMP-9, glioma-cell invasion was retarded compared with mock and EV/SV-treated groups, demonstrated by Matrigel-invasion assay and spheroid-invasion assay. Downregulation of uPA, uPAR and MMP-9 using RNAi inhibited angiogenesis in an in vitro (co-culture) model. Direct intratumoral injections of plasmid DNA expressing hpRNA for uPA, uPAR and MMP-9 significantly regressed pre-established intracranial tumors in nude mice. In addition, cells treated with RNAi for uPAR, uPA and MMP-9 showed reduced pERK levels compared with parental and EV/SV-treated SNB19 cells. Our results support the therapeutic potential of RNAi as a method for gene therapy in treating gliomas.     

Interferons inhibit tumor necrosis factor-alpha-mediated matrix metalloproteinase-9 activation via interferon regulatory factor-1 binding competition with NF-kappa B

Enhanced expression of matrix metalloproteinase-9 (MMP-9) correlates with invasion during tumor progression. Interferons (IFNs) inhibit MMP-9 activation in response to tumor necrosis factor-alpha (TNF-alpha), and the latter activates the MMP-9 gene through NF-kappaB. Understanding the molecular basis for MMP-9 inhibition may provide tools to control cell invasion. The data reported here show the critical role of interferon regulatory factor-1 (IRF1) in the inhibition of MMP-9. (i) IFN treatment suppresses TNF-alpha-induced MMP-9 reporter activity in STAT1(+/+) cells but not in STAT1(-/-) cells. (ii) IRF1 transfection blocks TNF-alpha-mediated MMP-9 activation. (iii) IFNs phosphorylate STAT1 and induce IRF1 but do not affect Ikappa-B degradation nor NF-kappaB nuclear translocation. (iv) Nuclear NF-kappaB (p50/p65) and IRF1, but not STAT1, bind to the MMP-9 promoter region containing an IFN-responsive-like element overlapping the NF-kappaB-binding site. (v) Recombinant IRF1, although unable to bind to an NF-kappaB consensus sequence, competes with NF-kappaB proteins for binding to the MMP-9 promoter. (vi) Conversely recombinant p50/p65 proteins reduce IRF1-DNA binding. (vii) In cells cotransfected with IRF1 and/or p65 expression vectors, an excess of IRF1 reduces MMP-9 reporter activity, whereas an excess of p65 blocks the inhibitory effect of IFN-gamma. Thus, in contrast to the known synergism between IRF1 and NF-kappaB, our data identify a novel role for IRF1 as a competitive inhibitor of NF-kappaB binding to the particular MMP-9 promoter context.     

Cytokine-induced upregulation of NF-kappaB, IL-8, and ICAM-1 is dependent on colonic cell polarity: implication for PKCdelta

As described for a long time, carcinoma-derived Caco-2 cells form a polarized epithelium in culture, whereas HT29-D4 cells are nonpolarized and undifferentiated but can form a polarized monolayer when cultured in a galactose-supplemented medium. Using NF-kappaB translocation and IL-8 and ICAM-1 gene activation as an index, we have studied the relationship between the differentiation state and the cell response to cytokines. We found that differentiated Caco-2 and HT29-D4 cells were responsive to both cytokines TNFalpha- and IL-1beta-mediated activation of NF-kappaB but that undifferentiated HT29-D4 cells were unresponsive to IL-1beta. However, the expression of endogenous ICAM-1 and IL-8 genes was upregulated by these cytokines in either cell lines differentiated or not. Upregulation of ICAM-1 gene occurred when IL-1beta or TNFalpha was added to the basal, but not apical surface of the differentiated epithelia. Finally, it appeared that in polarized HT29-D4 cells, the IL-1beta-induced translocation of NF-kappaB was connected to PKCdelta translocation.     

Matrix metalloproteinases and their endogenous regulators in squamous cervical carcinoma (A review of own results)

Own results of long-term studies of expression of matrix metalloproteinases (MMPs) and their endogenous regulators examined in fibroblasts transformed by oncogene E7 HPV16 (TF), immortalized fibroblasts (IF), cell lines associated with HPV16 and HPV18, and tumor tissue samples from patients with squamous cervical carcinoma (SCC) associated with HPV16 have been summarized. Transfection of fibroblasts with the E7 HPV16 oncogen was accompanied by induction of collagenase (MMP-1, MMP-14) and gelatinase (MMP-9) gene expression and the increase in catalytic activity of these MMP, while gelatinase MMP-2 expression remained unchanged. MMP expression correlated with the tumorigenic of transformed clones. Expression of MMP-9 was found only in TF. In TF expression mRNA TIMP-1 decreased, while expression of the genatinase inhibitor, TIMP-2, increased. Collagenase activity and expression of the MMP-14 (collagenase) mRNA increased, while gelatinase activity remained unchanged. The destructive potential of TF is associated with induction of collagenases, gelatinase MMP-9 and decreased levels of MMP inhibitors. MMP-9 may serve as a TF marker. Invasive potential of cell lines associated with HPV18 (HeLa and S4-1) was more pronounced than that of cell lines associated with HPV16 (SiHa and Caski). In most cell lines mRNA levels of collagenases MMP-1 and MMP-14 and the activator (uPA) increased, while gelatinase MMP-2 mRNA and tissue inhibitors mRNAs changed insignificantly. MMP-2 activity significantly increased in Caski and HeLa cell lines, while MMP-9 expression in these cell lines was not detected. The comparative study of expression MMP of and their endogenous regulators performed using SCC tumor samples associated with HPV16 has shown that the invasive and metastatic potentials of tumor tissue in SCC is obviously associated with increased expression of collagenases MMP-1, MMP-14 and gelatinase MMP-9, as well as decreased expression of inhibitors (TIMP-1 and TIMP-2), and to a lesser extent with increased expression of MMP-2. MMP-1 and MMP-9 can serve as markers of invasive and metastatic potential of the SCC tumor. The morphologically normal tissue adjacent to the tumor tissue is characterized by significant expression of MMP-1, MMP-2, and MMP-9. This also contributes to the increased destructive potential of the tumor.     

Arsenic trioxide (As2O3) inhibits invasion of HT1080 human fibrosarcoma cells: role of nuclear factor-kappaB and reactive oxygen species

In order to define the role of As2O3 in regulating the tumor cell invasiveness, the effects of As2O3 on secretion of matrix metalloproteinases (MMPs) and urokinase plasminogen activator (uPA), and in vitro invasion of HT1080 human fibrosarcoma cells were examined. As2O3 inhibited cell adhesion to the collagen matrix in a concentration dependent manner, whereas the same treatment enhanced cell to cell interaction. In addition, As2O3 inhibited migration and invasion of HT1080 cells stimulated with phorbol 12-myristate 13-aceate (PMA), and suppressed the expression of MMP-2, -9, membrane type-1 MMP, uPA, and uPA receptor (uPAR). In contrast, As2O3 increased the expression of tissue inhibitor of metalloproteinase (TIMP)-1 and PA inhibitor (PAI)-1, and reduced the MMP-2, -9, and uPA promoter activity in the presence and absence of PMA. Furthermore, the promoter stimulating and DNA binding activity of nuclear factor-kappaB (NF-kappaB) was blocked by As2O3, whereas the activator protein-1 activity was unchanged. Pretreatment of the cells with N-acetyl-L-cysteine (NAC) significantly prevented suppression of MMPs and uPA secretion, DNA binding activity of NF-kappaB, and in vitro invasion of HT1080 cells by As2O3, suggesting a role of reactive oxygen species (ROS) in this process. These results suggest that As2O3 inhibits tumor cell invasion by modulating the MMPs/TIMPs and uPA/uPAR/PAI systems of extracellular matrix (ECM) degradation. In addition, the generation of ROS and subsequent suppression of NF-kappaB activity by As2O3 might partly be responsible for the phenomena. Overall, As2O3 shows potent activity controlling tumor cell invasiveness in vitro.     

Distinct mechanism of Helicobacter pylori-mediated NF-kappa B activation between gastric cancer cells and monocytic cells

NF-kappaB is a critical regulator of genes involved in inflammation. Gastric epithelial cells and macrophages are considered the main sources of pro-inflammatory cytokines. We investigated NF-kappaB activation by Helicobacter pylori in MKN45 gastric epithelial cells and THP-1 monocytic cells. Although, cag pathogenicity island (PAI)-positive H. pylori (wild type) activated NF-kappaB in both cells, isogenic mutant of cagE (DeltacagE) activated it only in THP-1 cells. Supernatant from the wild type culture could activate NF-kappaB in THP-1 cells but not in MKN45 cells. High density cDNA array analysis revealed that mRNA expression of NF-kappaB-regulated genes such as interleukin (IL)-8, tumor necrosis factor-alpha (TNFalpha), and IL-1beta was significantly up-regulated by the wild type in both cells, whereas it was up-regulated by DeltacagE only in THP-1 cells. Experiments using CD14-neutralizing antibody and IL-1 receptor-associated kinase (IRAK) assay showed that both wild type and DeltacagE H. pylori activated NF-kappaB through CD14 and IRAK in THP-1 cells but not in MKN45 cells. Macrophages from C3H/HeJ mice carrying point mutation in the Toll-like receptor 4 (TLR4) gene showed decreased NF-kappaB activation and TNFalpha secretion compared with C3H/HeN mouse macrophage when treated with H. pylori. In conclusion, H. pylori-induced NF-kappaB activation in epithelial cells is dependent on cag PAI and contact but does not involve CD14 and IRAK, whereas in macrophage/monocytic cells it is independent of cag PAI or contact but involves CD14 and TLR4.     

Matrix metalloproteinases (MMP)—MMP-1,-2,-9 and endogenous regulators of their activity in cervical carcinoma cell lines transformed by the E7 oncogene HPV16 and HPV18

Matrix metalloproteinases (MMP) play a key role in development of tumor invasion and metastasis. The aim of the work was to elucidate peculiarities of expression of MMP-1, MMP-2, MMP-9 and regulators of their activity: plasminogen activator uPA and tissue inhibitors of MMPs TIMP-1 and TIMP-2 in human cell lines of squamous cell carcinoma (SCC).     

Interstitial collagenase, gelatinases A and B and their endogenous inhibitors in squamous cell cervical carcinomas

Interstitial collagenase and gelatinases are matrix metalloproteinases (MMP), which play the key role in tumor invasion and metastasis determining tumor malignancy. The aim of this study was to elucidate peculiarities of expression of interstitial collagenase (MMP-1), gelatinases A and B (MMP-2 and MMP-9) and their endogenous tissue inhibitors TIMP-1 and TIMP-2 as invasive factors of squamous cell carcinomas (SCC) of human cervical cancer. The study was carried out using 24 specimens of SCC and 11 specimens of morphologically normal tissue adjacent to tumor. All carcinoma specimens expressed the E7 HPV-16 gene. Results obtained indicate that the increased expression of MMP-1 and MMP-9 and low expression of TIMP-1 and TIMP-2 make the main contribution to the destructive (invasive) potential of SCC. Changes in MMP-2 expression are less important. In the specimens of morphologically normal tissue adjacent to the tumor, substantial expression of MMP-1, MMP-2 and MMP-9 was registered. This expression appears to make additional contribution to the tumor destructive potential.     

Inhibition by TNF-alpha and IL-4 of cationic lipid mediated gene transfer in cystic fibrosis tracheal gland cells

BACKGROUND: In vivo, tracheal gland serous cells highly express the cystic fibrosis transmembrane conductance regulator (cftr) gene. This gene is mutated in the lethal monogenic disease cystic fibrosis (CF). Clinical trials in which the human CFTR cDNA was delivered to the respiratory epithelia of CF patients have resulted in weak and transient gene expression. METHODS AND RESULTS: As CF is characterized by mucus inspissation, airway infection, and severe inflammation, we tested the hypothesis that inflammation and especially two cytokines involved in the Th1/Th2 inflammatory response, interleukin 4 (IL-4) and TNFalpha, could inhibit gene transfer efficiency using a model of human CF tracheal gland cells (CF-KM4) and Lipofectamine reagent as a transfection reagent. The specific secretory defects of CF-KM4 cells were corrected by Lipofectamine-mediated human CFTR gene transfer. However, this was altered when cells were pre-treated with IL-4 and TNFalpha. Inhibition of luciferase reporter gene expression by IL-4 and TNFalpha pre-treated CF-KM4 cells was measured by activity and real-time RT-PCR. Both cytokines induced similar and synergistic inhibition of transgene expression and activity. This cytokine-mediated inhibition could be prevented by anti-inflammatory agents such as glucocorticoids but not by non-steroidal (NSAI) agents. CONCLUSIONS: This data suggests that an inflammatory context generated by IL-4 and TNFalpha can inhibit human CFTR gene transfer in CF tracheal gland cells and that glucocorticoids may have a protecting action.     

Peroxisome proliferator-activated receptor-gamma in cystic fibrosis lung epithelium

The pathophysiology of cystic fibrosis (CF) inflammatory lung disease is not well understood. CF airway epithelial cells respond to inflammatory stimuli with increased production of proinflammatory cytokines as a result of increased NF-kappaB activation. Peroxisome proliferator-activated receptor-gamma (PPARgamma) inhibits NF-kappaB activity and is reported to be reduced in CF. If PPARgamma participates in regulatory dysfunction in the CF lung, perhaps PPARgamma ligands might be useful therapeutically. Cell models of CF airway epithelium were used to evaluate PPARgamma expression and binding to NF-kappaB at basal and under conditions of inflammatory stimulation by Pseudomonas aeruginosa or TNFalpha/IL-1beta. An animal model of CF was used to evaluate the potential of PPARgamma agonists as therapeutic agents in vivo. In vitro, PPARgamma agonists reduced IL-8 and MMP-9 release from airway epithelial cells in response to PAO1 or TNFalpha/IL-1beta stimulation. Less NF-kappaB bound to PPARgamma in CF than normal cells, in two different assays; PPARgamma agonists abrogated this reduction. PPARgamma bound less to its target DNA sequence in CF cells. To test the importance of the reported PPARgamma inactivation by phosphorylation, we observed that inhibitors of ERK, but not JNK, were synergistic with PPARgamma agonists in reducing IL-8 secretion. In vivo, administration of PPARgamma agonists reduced airway inflammation in response to acute infection with P. aeruginosa in CF, but not wild-type, mice. In summary, PPARgamma inhibits the inflammatory response in CF, at least in part by interaction with NF-kappaB in airway epithelial cells. PPARgamma agonists may be therapeutic in CF.