Pleiotropic actions of miR-21 highlight the critical role of deregulated stromal microRNAs during colorectal cancer progression.

The oncogene microRNA-21 (miRNA; miR-21) is overexpressed in most solid organ tumours; however, a recent examination of stage II colorectal cancer (CRC) specimens suggests this may be a stromal phenomenon and not only a feature of cancer cells. In vitro and in vivo studies show that miR-21 has potent pro-metastatic effects in various malignant carcinoma cell lines. The tumour microenvironment has also been identified as a key actor during the metastatic cascade; however to date the significance of deregulated miR-21 expression within the cancer-associated stroma has not been examined. In the present study, a quantitative RT-PCR-based analysis of laser microdissected tissue confirmed that miR-21 expression is associated with a four-fold mean increase in CRC stroma compared with normal tissue. In situ hybridisation using locked nucleic acid probes localised miR-21 expression predominantly to fibroblasts within tumour-associated stroma. To study the molecular and biological impact of deregulated stromal miR-21 in CRC, stable ectopic expression was induced in immortalised fibroblasts. This resulted in upregulated α-smooth muscle actin expression implying miR-21 overexpression is driving the fibroblast-to-myofibroblast transdifferentiation. Conditioned medium from miR-21-overexpressing fibroblasts protected CRC cells from oxaliplatin-induced apoptosis and increased their proliferative capacity. 3D organotypic co-cultures containing fibroblasts and CRC cells revealed that ectopic stromal miR-21 expression was associated with increased epithelial invasiveness. Reversion-inducing cysteine-rich protein with kazal motifs, an inhibitor of matrix-remodelling enzyme MMP2, was significantly downregulated by ectopic miR-21 in established and primary colorectal fibroblasts with a reciprocal rise in MMP2 activity. Inhibition of MMP2 abrogated the invasion-promoting effects of ectopic miR-21. This data, which characterises a novel pro-metastatic mechanism mediated by miR-21 in the CRC stroma, highlights the importance of miRNA deregulation within the tumour microenvironment and identifies a potential application for stromal miRNAs as biomarkers in cancer.

R-deprenyl: pharmacological spectrum of its activity.

Deprenyl has been discovered by Knoll and co-workers. The R-enantiomer of deprenyl (selegiline) is a selective and irreversible inhibitor of the B-isoform of monoamine oxidase (MAO-B) enzyme. Due to its dopamine potentiating and possible neuroprotective properties it has an established role in the treatment of parkinsonian patients. By inhibiting MAO-B enzyme, R-deprenyl decreases the formation of hydrogen peroxide, alleviating the oxidative stress also reduced by increased expression of antioxidant enzymes (superoxide dismutases and catalase) reported during chronic treatment. It was shown to prevent the detrimental effects of neurotoxins like MPTP and DSP-4. R-Deprenyl elicits neuroprotective and neuronal rescue activities in concentrations too low to inhibit MAO-B. It is extensively metabolized and some of the metabolites possess pharmacological activities, thus their contribution to neuroprotective properties was also suggested. The recently identified deprenyl-N-oxide is extensively studied in our laboratory. Effects other than neuroprotection, like influencing cell adhesion and proliferation cannot be neglected.

Upregulation of Eps8 in oral squamous cell carcinoma promotes cell migration and invasion through integrin-dependent Rac1 activation.

Oral squamous cell carcinoma (OSCC) is a lethal disease and early death usually occurs as a result of local invasion and regional lymph node metastases. Current treatment regimens are, to a certain degree, inadequate, with a 5-year mortality rate of around 50% and novel therapeutic targets are urgently required. Using expression microarrays, we identified the eps8 gene as being overexpressed in OSCC cell lines relative to normal oral keratinocytes, and confirmed these findings using RT-PCR and western blotting. In human tissues, we found that Eps8 was upregulated in OSCC (32% of primary tumors) compared with normal oral mucosa, and that expression correlated significantly with lymph node metastasis (P=0.032), suggesting a disease-promoting effect. Using OSCC cell lines, we assessed the functional role of Eps8 in tumor cells. Although suppression of Eps8 produced no effect on cell proliferation, both cell spreading and migration were markedly inhibited. The latter cell functions may be modulated through the small GTP-ase, Rac1 and we used pull-down assays to investigate the role of Eps8 in Rac1 signaling. We found that alphavbeta6- and alpha5beta1-integrin-dependent activation of Rac1 was mediated through Eps8. Knockdown of either Eps8 or Rac1, inhibited integrin-dependent cell migration similarly and transient expression of constitutively active Rac1 restored migration of cells in which Eps8 expression had been suppressed. We also showed that knockdown of Eps8 inhibited tumor cell invasion in an organotypic model of OSCC. These data suggest that Eps8 and Rac1 are part of an integrated signaling pathway modulating integrin-dependent tumour cell motility and identify Eps8 as a possible therapeutic target.

Deprenyl: from chemical synthesis to neuroprotection.

During the last decades (-)-deprenyl has become the golden standard of MAO-B inhibitors. It possesses dopamine potentiating and antioxidant properties; however, its effects cannot be explained solely by the enzyme inhibitory action. (-)-Deprenyl prevents the toxicity of certain selective neurotoxins and recently it was demonstrated to increase cell-cell adhesion as well. The complexity of its pharmacological effects reflects the action of both the parent compound and the active metabolites. (-)-Deprenyl and related propargylamines (DRPs) show neuroprotective features in a variety of in vitro and in vivo models that is dependent on the propargyl moiety. The main presumptive targets to date include glyceraldehyde-3-phosphate dehydrogenase, poly(ADP-ribose) polymerase, some kinase cascades, as well as pro- and antiapoptotic proteins, beside the inhibition of MAO-B. The antiapoptotic activity of DRPs converges upon the maintenance of mitochondrial integrity, due to the initiation of a complex transcriptional program, the details of which are yet to be elucidated.

Oxidative stress and non-enzymatic glycation in IgA nephropathy.

AIM: Approximately 20-50% of IgA nephropathy patients develop end-stage renal disease. We have previously found enhanced oxidative stress and decreased antioxidant capacity in red blood cells of IgA nephropathy patients. In this study we assess oxidative stress, non-enzymatic glycation, oxidative resistance of low-density lipoprotein and its alpha-tocopherol content in these patients. PATIENTS AND METHODS: Non-enzymatic glycation and oxidative stress were assessed in 88 IgA nephropathy patients by measuring advanced glycation end products, Nepsilon-carboxymethyl-lysine, thiobarbituric acid reactive substances, oxidative resistance of low-density lipoprotein and its alpha-tocopherol content. RESULTS: Advanced glycation end products (2659 +/- 958 a.u.) and Nepsilon-carboxymethyl-lysine (563 +/- 215 ng/ml) were significantly higher in IgA nephropathy patients with decreased renal function compared to those with normal renal function (p < 0.002) or controls (p < 0.001). Thiobarbituric acid-reactive substances in plasma and associated with low-density lipoprotein were significantly elevated and oxidative resistance of low-density lipoprotein was significantly reduced in all groups of IgA nephropathy patients. There was no significant difference in circulating fluorescent advanced glycation end products, Nepsilon-carboxymethyl-lysine, thiobarbituric acid-reactive substances levels, oxidative resistance of low-density lipoprotein and its alpha-tocopherol content between patients with normal vs. impaired glucose metabolism. Low alpha-tocopherol content of low-density lipoprotein was accompanied with decreased oxidative resistance, depletion in polyunsaturated fatty acids, elevated saturated fatty acids and thiobarbituric acid-reactive substances within low-density lipoprotein suggesting enhanced lipid peroxidation. CONCLUSIONS: Decreased oxidative resistance of low-density lipoprotein and enhanced oxidative stress are common features in IgA nephropathy, while increased non-enzymatic glycation occurs as renal function declines.

Increased cell-cell adhesion, a novel effect of R-(-)-deprenyl.

The neuroprotective effect of the antiparkinsonian monoamine oxidase (MAO)-B inhibitor, R-(-)-deprenyl has been under investigation for years. Cytoskeleton, a main component of cell adhesion, is involved in the development of R-(-)-deprenyl-responsive diseases, the effect of the drug on cell adhesion, however, is not known. We examined the effect of R-(-)-deprenyl on cell-cell adhesion of neuronal and non-neuronal cells. R-(-)-deprenyl treatment resulted in a cell type- and concentration-dependent increase in cell-cell adhesion of PC12 and NIH3T3 cells at concentrations lower than those required for MAO-B inhibition, while S-(+)-deprenyl was not effective. This acitvity of R-(-)-deprenyl was not prevented by the cytochrome P-450 inhibitor, SKF525A, while deprenyl-N-oxide, a newly described metabolite, also induced an increase in cell-cell adhesion. The effect of R-(-)-deprenyl was not reversible during a 24-hour recovery period. In summary, we described a new, MAO-B independent effect of R-(-)-deprenyl on cell-cell adhesion which can contribute to its neuroprotective function.