Recent studies are reviewed indicating that the transcription factor early growth response-1 (Egr1) is a direct regulator of multiple tumor suppressors including TGFb1, PTEN, p53, and fibronectin. The downstream pathways of these factors display multiple nodes of interaction with each other, suggesting the existence of a functional network of suppressor factors that serve to maintain normal growth regulation and resist the emergence of transformed variants. Paradoxically, Egr1 is oncogenic in prostate cancer. In the majority of these cancers, PTEN or p53 is inactive. It is suggested that these defects in the suppressor network allow for the unopposed induction of TGFb1 and fibronectin, which favor transformation and survival of prostate tumor epithelial cells, and explain the role of Egr1 in prostate cancer. Egr1 is a novel and logical target for intervention by gene therapy methods, and targeting methods are discussed.

Transcription factor early growth response-1 (Egr-1) is a crucial regulator of cell growth, differentiation and survival. Several observations suggest that Egr-1 is growth promoting in prostate cancer cells and that blocking its function may impede cancer progression. To test this hypothesis, we developed phosphorothioate antisense oligonucleotides that efficiently inhibit Egr-1 expression without altering the expression of other family members Egr-2, Egr-3 and Egr-4. In TRAMP mouse-derived prostate cancer cell lines, our optimal antisense oligonucleotide decreased the expression of the Egr-1 target gene transforming growth factor-b1 whereas a control oligonucleotide had no effect, indicating that the antisense blocked Egr-1 function as a transcription factor. The antisense oligonucleotide deregulated cell cycle progression and decreased proliferation of the three TRAMP cell lines by an average of 5473%. Both colony formation and growth in soft agar were inhibited by the antisense oligonucleotide. When TRAMP mice were treated systemically for 10 weeks, the incidence of palpable tumors at 32 weeks of age in untreated mice or mice injected with the control scramble oligonucleotide was 87%, whereas incidence of tumors in antisense-Egr-1-treated mice was significantly reduced to 37% (P ¼ 0.026). Thus, Egr-1 plays a functional role in the transformed phenotype and may represent a valid target for prostate cancer therapy.

Muscarinic receptors, expressed in several primary and metastatic tumours, appear to be implicated in their growth and propagation. In this work we have demonstrated that M2 muscarinic receptors are expressed in glioblastoma human specimens and in glioblastoma cell lines. Moreover, we have characterized the effects of the M2 agonist arecaidine on cell growth and survival both in two different glioblastoma cell lines (U251MG and U87MG) and in primary cultures obtained from different human biopsies. Cell growth analysis has demonstrated that the M2 agonist arecaidine strongly decreased cell proliferation in both glioma cell lines and primary cultures. This effect was dose and time dependent. FACS analysis has confirmed cell cycle arrest at G1/S and at G2/M phase in U87 cells and U251 respectively. Cell viability analysis has also shown that arecaidine induced severe apoptosis, especially in U251 cells. Chemosensitivity assays have, moreover, shown arecaidine and temozolomide similar effects on glioma cell lines, although IC50 value for arecaidine was significantly lower than temozolomide. In conclusion, we report for the first time that M2 receptor activation has a relevant role in the inhibition of glioma cell growth and survival, suggesting that M2 may be a new interesting therapeutic target to investigate for glioblastoma therapy.

Multifunctional nanomaterials with the ability to respond to near-infrared (NIR) light stimulation are vital for the development of highly efficient biomedical nanoplatforms with a polytherapeutic approach. Inspired by the mesoglea structure of jellyfish bells, a biomimetic multifunctional nanostructured pillow with fast photothermal responsiveness for NIR light-controlled on-demand drug delivery is developed. We fabricate a nanoplatform with several hierarchical levels designed to generate a series of controlled, rapid, and reversible cascade-like structural changes upon NIR light irradiation. The mechanical contraction of the nanostructured platform, resulting from the increase of temperature to 42 °C due to plasmonic hydrogel–light interaction, causes a rapid expulsion of water from the inner structure, passing through an electrospun membrane anchored onto the hydrogel core. The mutual effects of the rise in temperature and water flow stimulate the release of molecules from the nanofibers. To expand the potential applications of the biomimetic platform, the photothermal responsiveness to reach the typical temperature level for performing photothermal therapy (PTT) is designed. The on-demand drug model penetration into pig tissue demonstrates the efficiency of the nanostructured platform in the rapid and controlled release of molecules, while the high biocompatibility confirms the pillow potential for biomedical applications based on the NIR light-driven multitherapy strategy.

Glial cell proliferation in culture is under the control of metabotropic glutamate (mGlu) receptors. We have examined whether this control extends to human glioma cells. Primary cultures were prepared from surgically removed human glioblastomas. RT-PCR combined with western blot analysis showed that most of the cultures (eight out of 11) expressed group-II mGlu receptors. In two selected cultures (MZC-12 and FCN-9), the mGlu2/3 receptor antagonist, LY341495, slowed cell proliferation when applied to the growth medium from the second day after plating. This effect was reversible because linear cell growth was restored after washing out the drug. LY341495 reduced glioma cell proliferation at concentrations lower than 100 nM, which are considered as selective for mGlu2/3 receptors. In addition, its action was mimicked by the putative mGlu2/3 receptor antagonist (2S)-a-ethylglutamate. The anti-proliferative effect of LY341495 was confirmed by measuring [methyl-3 H]-thymidine incorporation in cultures arrested in G 0 phase of the cell cycle and then stimulated to proliferate by the addition of 10% fetal calf serum or 100 ng/mL of epidermal growth factor (EGF). In cultures treated with EGF, LY341495 was also able to reduce the stimulation of the mitogen-activated protein kinase (MAPK) pathway, as well as the induction of cyclin D1. Both effects, as well as decreased [methyl-3 H]-thymidine incorporation, were partially reduced by co-addition of the potent mGlu2/3 receptor agonist, LY379268. We conclude that activation of group-II mGlu receptors supports the growth of human glioma cells in culture and that antagonists of these receptors should be tested for their ability to reduce tumour growth in vivo.

Dual-specificity phosphatase 6 (DUSP6, mitogen-activated protein kinase (MAPK) phosphatase 3 or PYST1) dephosphorylates phosphotyrosine and phosphothreonine residues on extracellular signal-regulated kinase (ERK1/ 2; MAPK1/2) to inactivate the ERK1/2 kinase. DUSP6 is a critical regulator of the ERK signaling cascade and has been implicated as a tumor suppressor. We report here experimental evidences that DUSP6 is transcriptionally upregulated in primary and long-term cultures of human glioblastoma, as assayed by northern hybridization and real-time quantitative PCR, producing constitutive high level of protein expression. Functional assays were performed with adenovirus-mediated expression of DUSP6 in glioblastoma cultures. Protein overexpression inhibits growth by inducing G1-phase delay and increased mitogenic/anchorage dependence and clonogenic potential in vitro. Changes in cell morphology were associated with an increased tumor growth in vivo. Chemoresistance is a major cause of treatment failure and poor outcome in human glioblastomas. Importantly, DUSP6 overexpression increased resistance to cisplatinmediated cell death in vitro and in vivo. Antisense-mediated depletion of DUSP6 acted in lowering the threshold to anticancer DNA-damaging drugs. We conclude that upregulation of DUSP6 exerts a tumor-promoting role in human glioblastomas exacerbating the malignant phenotype.

Glioblastoma cells in vivo are exposed to a variety of promigratory signals, including undefined serum components that infiltrate into high grade gliomas as result of blood-brain barrier breakdown. Glioblastoma cell migration has been further shown to depend heavily on ion channels activity. We have then investigated the modulatory effects of fetal calf serum (FCS) on ion channels, and their involvement in U87-MG cells migration. Using the perforated patch-clamp technique we have found that, in a subpopulation of cells (42%), FCS induced: (1) an oscillatory activity of TRAM-34 sensitive, intermediate-conductance calcium-activated K (IK(Ca) ) channels, mediated by calcium oscillations previously shown to be induced by FCS in this cell line; (2) a stable activation of a DIDS- and NPPB-sensitive Cl current displaying an outward rectifying instantaneous current-voltage relationship and a slow, voltage-dependent inactivation. By contrast, in another subpopulation of cells (32%) FCS induced a single, transient IK(Ca) current activation, always accompanied by a stable activation of the Cl current. The remaining cells did not respond to FCS. In order to understand whether the FCS-induced ion channel activities are instrumental to promoting cell migration, we tested the effects of TRAM-34 and DIDS on the FCS-induced U87-MG cell migration using transwell migration assays. We found that these inhibitors were able to markedly reduce U87-MG cell migration in the presence of FCS, and that their co-application resulted in an almost complete arrest of migration. It is concluded that the modulation of K and Cl ion fluxes is essential for the FCS-induced glioblastoma cell migration.

The epithelial-to-mesenchymal transition (EMT) is an essential trans-differentiation process, which plays a critical role in embryonic development, wound healing, tissue regeneration, organ fibrosis, and cancer progression. It is the fundamental mechanism by which epithelial cells lose many of their characteristics while acquiring features typical of mesenchymal cells, such as migratory capacity and invasiveness. Depending on the contest, EMT is complemented and balanced by the reverse process, the mesenchymal-to-epithelial transition (MET). In the saving economy of the living organisms, the same (Ying-Yang) tool is integrated as a physiological strategy in embryonic development, as well as in the course of reparative or disease processes, prominently fibrosis, tumor invasion and metastasis. These mechanisms and their related signaling (e.g., TGF-β and BMPs) have been effectively studied in vitro by tissue-derived cell spheroids models. These three-dimensional (3D) cell culture systems, whose phenotype has been shown to be strongly dependent on TGF-β-regulated EMT/MET processes, present the advantage of recapitulating in vitro the hypoxic in vivo micro-environment of tissue stem cell niches and their formation. These spheroids, therefore, nicely reproduce the finely regulated Ying-Yang equilibrium, which, together with other mechanisms, can be determinant in cell fate decisions in many pathophysiological scenarios, such as differentiation, fibrosis, regeneration, and oncogenesis. In this review, current progress in the knowledge of signaling pathways affecting EMT/MET and stemness regulation will be outlined by comparing data obtained from cellular spheroids systems, as ex vivo niches of stem cells derived from normal and tumoral tissues. The mechanistic correspondence in vivo and the possible pharmacological perspective will be also explored, focusing especially on the TGF-β-related networks, as well as others, such as SNAI1, PTEN, and EGR1. This latter, in particular, for its ability to convey multiple types of stimuli into relevant changes of the cell transcriptional program, can be regarded as a heterogeneous "stress-sensor" for EMT-related inducers (growth factor, hypoxia, mechano-stress), and thus as a therapeutic target.