MiR-128 up-regulation inhibits Reelin and DCX expression and reduces neuroblastoma cell motility and invasiveness

MicroRNAs are a class of sophisticated regulators of gene expression, acting as post-transcriptional inhibitors that recognize their target mRNAs through base pairing with short regions along the 3'UTRs. Several microRNAs are tissue specific, suggesting a specialized role in tissue differentiation or maintenance, and quite a few are critically involved in tumorigenesis. We studied miR-128, a brain-enriched microRNA, in retinoic acid-differentiated neuroblastoma cells, and we found that this microRNA is up-regulated in treated cells, where it down-modulates the expression of two proteins involved in the migratory potential of neural cells: Reelin and DCX. Consistently, miR-128 ectopic overexpression suppressed Reelin and DCX, whereas the LNA antisense-mediated miR-128 knockdown caused the two proteins to increase. Ectopic miR-128 overexpression reduced neuroblastoma cell motility and invasiveness, and impaired cell growth. Finally, the analysis of a small series of primary human neuroblastomas showed an association between high levels of miR-128 expression and favorable features, such as favorable Shimada category or very young age at diagnosis. Thus, we provide evidence for a role for miR-128 in the molecular events modulating neuroblastoma progression and aggressiveness

The inhibition of the highly expressed miR-221 and miR-222 impairs the growth of prostate carcinoma xenografts in mice

MiR-221 and miR-222 are two highly homologous microRNAs whose upregulation has been recently described in several types of human tumors, for some of which their oncogenic role was explained by the discovery of their target p27, a key cell cycle regulator. We previously showed this regulatory relationship in prostate carcinoma cell lines in vitro, underlying the role of miR-221/222 as inducers of proliferation and tumorigenicity

An anti-VEGF ribozyme embedded within the adenoviral VAI sequence inhibits glioblastoma cell angiogenic potential in vitro

Vascular endothelial growth factor (VEGF) plays an important role in tumor angiogenesis, where it functions as one of the major angiogenic factors sustaining growth and draining catabolites. In this study, we developed an anti-VEGF ribozyme targeted to the 5' part of human VEGF mRNA. We endowed this ribozyme with an additional feature expected to improve its activity in vivo, by cloning it into a VAI transcriptional cassette. VAI is originally part of the adenovirus genome, and is characterized by high transcription rates, good stability due to its strong secondary structure and cytoplasmic localization. Transfection of U87 human glioblastoma cells with plasmid vectors encoding for this ribozyme resulted in a strong (-56%) reduction of VEGF secreted in the extracellular medium, indicating a good biological activity of the ribozyme. Moreover, this reduction in VEGF secretion had the important functional consequence of drastically diminishing the formation of tube-like structures of human umbilical vascular endothelial cells in a Matrigel in vitro angiogenesis assay. In conclusion, our VAI-embedded anti-VEGF ribozyme is a good inhibitor of angiogenesis in vitro, in a glioblastoma cell context. Thus, it may represent a useful tool for future applications in vivo, for antiangiogenic gene therapy of glioblastoma and of highly vascularized tumors

CoCl(2)-simulated hypoxia in skeletal muscle cell lines: Role of free radicals in gene up-regulation and induction of apoptosis

Since it was suggested that cobalt chloride (CoCl(2)) could mimic the O(2) sensing role of mitochondria by increasing reactive oxygen species (ROS) generation during normoxia, we studied the correlation between CoCl(2)-generation of free radicals and the induction of a hypoxic cellular response in myogenic cell lines. In both L6C5 and C2C12 cell lines, exposure to CoCl(2) induced an increase of intracellular oxidants, the accumulation of HIF-1alpha protein, and the expression of vascular endothelial growth factor (VEGF) and/or iNOS genes. On the other hand, only ascorbic acid, but not trolox, was effective in lowering the CoCl(2) gene up-regulation. Neither the cytotoxicity nor the apoptosis induced by CoCl(2) in skeletal muscle cells were modified by culture supplementation with either ascorbic acid or trolox. Thus, CoCl(2) treatment of myogenic cell lines may represent a useful and convenient in vitro model to study gene modulation induced by hypoxia in skeletal muscle, although cellular loss induced by this metal may involve mechanisms other than HIF-1alpha stabilization. It is unlikely, however, that ROS would represent the main mediators of CoCl(2) effects on muscle cells

Cellular responses to H(2)O(2) and bleomycin-induced oxidative stress in L6C5 rat myoblasts

In muscle cells, reactive oxygen species (ROS) are continually generated. It is believed that these molecules have a well-established role as physiological modulators of skeletal muscle functions, ranging from development to metabolism and from blood flow to contractile functions. Moreover, ROS may contribute to the development of muscle fatigue, inflammation, and degeneration, and may be implicated in many muscle diseases. The aim of the present study was to verify the role of short or prolonged exposure to oxidative stress, generated by different concentrations of H(2)O(2), on growth, chromosomal aberrations, and apoptosis induced in cultured L6C5 rat muscle cells used as model for myoblasts. Our results indicate that, in L6C5 cells, reactive oxygen intermediates (ROI) can activate distinct cell pathways leading to cell growth induction and development of resistant phenotype, or to chromosomal aberrations, cell cycle arrest, or cell death. The positive vs. negative effects of H(2)O(2)-altered redox potential in myoblasts are strictly related to the intensity of oxidative stress, likely depending on the types and number of cellular targets involved. Among these, DNA molecules appear to be very sensitive to breakage by H(2)O(2), although DNA damage is not directly responsible for ROI-induced apoptosis in L6C5 rat myoblasts

Vector-based RNA interference against vascular endothelial growth factor-A significantly limits vascularization and growth of prostate cancer in vivo

RNA interference technology is emerging as a very potent tool to obtain a cellular knockdown of a desired gene. In this work we used vector-based RNA interference to inhibit vascular endothelial growth factor (VEGF) expression in prostate cancer in vitro and in vivo. We demonstrated that transduction with a plasmid carrying a small interfering RNA targeting all isoforms of VEGF, dramatically impairs the expression of this growth factor in the human prostate cancer cell line PC3. As a consequence, PC3 cells loose their ability to induce one of the fundamental steps of angiogenesis, namely the formation of a tube-like network in vitro. Most importantly, our "therapeutic" vector is able to impair tumor growth rate and vascularization in vivo. We show that a single injection of naked plasmid in developing neoplastic mass significantly decreases microvessel density in an androgen-refractory prostate xenograft and is able to sustain a long-term slowing down of tumor growth. In conclusion, our results confirm the basic role of VEGF in the angiogenic development of prostate carcinoma, and suggest that the use of our vector-based RNA interference approach to inhibit angiogenesis could be an effective tool in view of future gene therapy applications for prostate cancer

Control of neoplastic cell proliferation and differentiation by restoration of 4-hydroxynonenal physiological concentrations

Several studies point to the existence of an inverse correlation between cellular lipid peroxidation and both cell proliferation and neoplastic transformation. In anaplastic cell lines products of membrane lipid peroxidation are very low or undetectable. Furthermore numerous results demonstrate effect of lipid peroxidation products on central biochemical pathways and intracellular signalling at physiological concentrations. 4-hydroxynonenal (HNE) is one of the most active products of lipid peroxidation. The restoration of HNE physiological concentrations in neoplastic cells may inhibit cell proliferation and modulate cell re-differentiation. This review try to summarize and critically discuss the effects of physiological concentrations of HNE on normal and neoplastic cell line