Inhibition of chloride intracellular channel 1 (CLIC1) as biguanide class-effect to impair human glioblastoma stem cell viability

The antidiabetic biguanide metformin exerts antiproliferative effects in different solid tumors. However, during preclinical studies, metformin concentrations required to induce cell growth arrest were invariably within the mM range, thus difficult to translate in a clinical setting. Consequently, the search for more potent metformin derivatives is a current goal for new drug development. Although several cell-specific intracellular mechanisms contribute to the anti-tumor activity of metformin, the inhibition of the chloride intracellular channel 1 activity (CLIC1) at G1/S transition is a key events in metformin antiproliferative effect in glioblastoma stem cells (GSCs). Here we tested several known biguanide-related drugs for the ability to affect glioblastoma (but not normal) stem cell viability, and in particular: phenformin, a withdrawn antidiabetic drug; moroxydine, a former antiviral agent; and proguanil, an antimalarial compound, all of them possessing a linear biguanide structure as metformin; moreover, we evaluated cycloguanil, the active form of proguanil, characterized by a cyclized biguanide moiety. All these drugs caused a significant impairment of GSC proliferation, invasiveness, and self-renewal reaching IC50values significantly lower than metformin, (range 0.054-0.53 mM vs. 9.4 mM of metformin). All biguanides inhibited CLIC1-mediated ion current, showing the same potency observed in the antiproliferative effects, with the exception of proguanil which was ineffective. These effects were specific for GSCs, since no (or little) cytotoxicity was observed in normal umbilical cord mesenchymal stem cells, whose viability was not affected by metformin and moroxydine, while cycloguanil and phenformin induced toxicity only at much higher concentrations than required to reduce GSC proliferation or invasiveness. Conversely, proguanil was highly cytotoxic also for normal mesenchymal stem cells. In conclusion, the inhibition of CLIC1 activity represents a biguanide class-effect to impair GSC viability, invasiveness, and self-renewal, although dissimilarities among different drugs were observed as far as potency, efficacy and selectivity as CLIC1 inhibitors. Being CLIC1 constitutively active in GSCs, this feature is relevant to grant the molecules with high specificity toward GSCs while sparing normal cells. These results could represent the basis for the development of novel biguanidestructured molecules, characterized by high antitumor efficacy and safe toxicological profile

Chloride intracellular channel 1 activity is not required for glioblastoma development but its inhibition dictates glioma stem cell responsivity to novel biguanide derivatives

Background: Chloride intracellular channel-1 (CLIC1) activity controls glioblastoma proliferation. Metformin exerts antitumor effects in glioblastoma stem cells (GSCs) inhibiting CLIC1 activity, but its low potency hampers its translation in clinical settings. Methods: We synthesized a small library of novel biguanide-based compounds that were tested as antiproliferative agents for GSCs derived from human glioblastomas, in vitro using 2D and 3D cultures and in vivo in the zebrafish model. Compounds were compared to metformin for both potency and efficacy in the inhibition of GSC proliferation in vitro (MTT, Trypan blue exclusion assays, and EdU labeling) and in vivo (zebrafish model), migration (Boyden chamber assay), invasiveness (Matrigel invasion assay), self-renewal (spherogenesis assay), and CLIC1 activity (electrophysiology recordings), as well as for the absence of off-target toxicity (effects on normal stem cells and toxicity for zebrafish and chick embryos). Results: We identified Q48 and Q54 as two novel CLIC1 blockers, characterized by higher antiproliferative potency than metformin in vitro, in both GSC 2D cultures and 3D spheroids. Q48 and Q54 also impaired GSC self-renewal, migration and invasion, and displayed low systemic in vivo toxicity. Q54 reduced in vivo proliferation of GSCs xenotransplanted in zebrafish hindbrain. Target specificity was confirmed by recombinant CLIC1 binding experiments using microscale thermophoresis approach. Finally, we characterized GSCs from GBMs spontaneously expressing low CLIC1 protein, demonstrating their ability to grow in vivo and to retain stem-like phenotype and functional features in vitro. In these GSCs, Q48 and Q54 displayed reduced potency and efficacy as antiproliferative agents as compared to high CLIC1-expressing tumors. However, in 3D cultures, metformin and Q48 (but not Q54) inhibited proliferation, which was dependent on the inhibition dihydrofolate reductase activity. Conclusions: These data highlight that, while CLIC1 is dispensable for the development of a subset of glioblastomas, it acts as a booster of proliferation in the majority of these tumors and its functional expression is required for biguanide antitumor class-effects. In particular, the biguanide-based derivatives Q48 and Q54, represent the leads to develop novel compounds endowed with better pharmacological profiles than metformin, to act as CLIC1-blockers for the treatment of CLIC1-expressing glioblastomas, in a precision medicine approach

The chemokine receptor CXCR2 and coronavirus-induced neurologic disease.

Inoculation with the neurotropic JHM strain of mouse hepatitis virus (MHV) into the central nervous system (CNS) of susceptible strains of mice results in an acute encephalomyelitis in which virus preferentially replicates within glial cells while excluding neurons. Control of viral replication during acute disease is mediated by infiltrating virus-specific T cells via cytokine secretion and cytolytic activity, however sterile immunity is not achieved and virus persists resulting in chronic neuroinflammation associated with demyelination. CXCR2 is a chemokine receptor that upon binding to specific ligands promotes host defense through recruitment of myeloid cells to the CNS as well as protecting oligodendroglia from cytokine-mediated death in response to MHV infection. These findings highlight growing evidence of the diverse and important role of CXCR2 in regulating neuroinflammatory diseases

The inhibition of FGF receptor 1 activity mediates sorafenib-induced antiproliferative effects in human mesothelioma tumor-initiating cells

Tumor-initiating cells (TICs), the subset of cells within tumors endowed with stem-like features, being highly resistant to conventional cytotoxic drugs, are the major cause of tumor relapse. The identification of molecules able to target TICs remains a significant challenge in cancer therapy. Using TIC-enriched cultures (MM1, MM3 and MM4), from 3 human malignant pleural mesotheliomas (MPM), we tested the effects of sorafenib on cell survival and the intracellular mechanisms involved. Sorafenib inhibited cell-cycle progression in all the TIC cultures, but only in MM3 and MM4 cells this effect was associated with induction of apoptosis via the down-regulation of Mcl-1. Although sorafenib inhibits the activity of several tyrosine kinases, its effects are mainly ascribed to Raf inhibition. To investigate the mechanisms of sorafenib-mediated antiproliferative activity, TICs were treated with EGF or bFGF causing, in MM3 and MM4 cells, MEK, ERK1/2, Akt and STAT3 phosphorylation. These effects were significantly reduced by sorafenib in bFGF-treated cells, while a slight inhibition occurred after EGF stimulation, suggesting that sorafenib effects are mainly due to FGFR inhibition. Indeed, FGFR1 phosphorylation was inhibited by sorafenib. A different picture was observed in MM1 cells, which, releasing high levels of bFGF, showed an autocrine activation of FGFR1 and a constitutive phosphorylation/activation of MEK-ERK1/2. A powerful inhibitory response to sorafenib was observed in these cells, indirectly confirming the central role of sorafenib as FGFR inhibitor. These results suggest that bFGF signaling may impact antiproliferative response to sorafenib of MPM TICs, which is mainly mediated by a direct FGFR targeting

Chemokines in cerebrospinal fluid correlate with cerebral metabolite patterns in HIV-infected individuals

Chemokines influence HIV neuropathogenesis by affecting the HIV life cycle, trafficking of macrophages into the nervous system, glial activation, and neuronal signaling and repair processes; however, knowledge of their relationship to in vivo measures of cerebral injury is limited. The primary objective of this study was to determine the relationship between a panel of chemokines in cerebrospinal fluid (CSF) and cerebral metabolites measured by proton magnetic resonance spectroscopy (MRS) in a cohort of HIV-infected individuals. One hundred seventy-one stored CSF specimens were assayed from HIV-infected individuals who were enrolled in two ACTG studies that evaluated the relationship between neuropsychological performance and cerebral metabolites. Concentrations of six chemokines (fractalkine, IL-8, IP-10, MCP-1, MIP-1β, and SDF-1) were measured and compared with cerebral metabolites individually and as composite neuronal, basal ganglia, and inflammatory patterns. IP-10 and MCP-1 were the chemokines most strongly associated with individual cerebral metabolites. Specifically, (1) higher IP-10 levels correlated with lower N-acetyl aspartate (NAA)/creatine (Cr) ratios in the frontal white matter and higher MI/Cr ratios in all three brain regions considered and (2) higher MCP-1 levels correlated with lower NAA/Cr ratios in frontal white matter and the parietal cortex. IP-10, MCP-1, and IL-8 had the strongest associations with patterns of cerebral metabolites. In particular, higher levels of IP-10 correlated with lower neuronal pattern scores and higher basal ganglia and inflammatory pattern scores, the same pattern which has been associated with HIV-associated neurocognitive disorders (HAND). Subgroup analysis indicated that the effects of IP-10 and IL-8 were influenced by effective antiretroviral therapy and that memantine treatment may mitigate the neuronal effects of IP-10. This study supports the role of chemokines in HAND and the validity of MRS as an assessment tool. In particular, the findings identify relationships between the immune response—particularly an interferon-inducible chemokine, IP-10—and cerebral metabolites and suggest that antiretroviral therapy and memantine modify the impact of the immune response on neurons

The SDF-1α/CXCR4 Axis is Required for Proliferation and Maturation of Human Fetal Pancreatic Endocrine Progenitor Cells

The chemokine receptor CXCR4 and ligand SDF-1α are expressed in fetal and adult mouse islets. Neutralization of CXCR4 has previously been shown to diminish ductal cell proliferation and increase apoptosis in the IFNγ transgenic mouse model in which the adult mouse pancreas displays islet regeneration. Here, we demonstrate that CXCR4 and SDF-1α are expressed in the human fetal pancreas and that during early gestation, CXCR4 colocalizes with neurogenin 3 (ngn3), a key transcription factor for endocrine specification in the pancreas. Treatment of islet like clusters (ICCs) derived from human fetal pancreas with SDF-1α resulted in increased proliferation of epithelial cells in ICCs without a concomitant increase in total insulin expression. Exposure of ICCs in vitro to AMD3100, a pharmacological inhibitor of CXCR4, did not alter expression of endocrine hormones insulin and glucagon, or the pancreatic endocrine transcription factors PDX1, Nkx6.1, Ngn3 and PAX4. However, a strong inhibition of β cell genesis was observed when in vitro AMD3100 treatment of ICCs was followed by two weeks of in vivo treatment with AMD3100 after ICC transplantation into mice. Analysis of the grafts for human C-peptide found that inhibition of CXCR4 activity profoundly inhibits islet development. Subsequently, a model pancreatic epithelial cell system (CFPAC-1) was employed to study the signals that regulate proliferation and apoptosis by the SDF-1α/CXCR4 axis. From a selected panel of inhibitors tested, both the PI 3-kinase and MAPK pathways were identified as critical regulators of CFPAC-1 proliferation. SDF-1α stimulated Akt phosphorylation, but failed to increase phosphorylation of Erk above the high basal levels observed. Taken together, these results indicate that SDF-1α/CXCR4 axis plays a critical regulatory role in the genesis of human islets

Neuronal Chemokines: Versatile Messengers In Central Nervous System Cell Interaction

Whereas chemokines are well known for their ability to induce cell migration, only recently it became evident that chemokines also control a variety of other cell functions and are versatile messengers in the interaction between a diversity of cell types. In the central nervous system (CNS), chemokines are generally found under both physiological and pathological conditions. Whereas many reports describe chemokine expression in astrocytes and microglia and their role in the migration of leukocytes into the CNS, only few studies describe chemokine expression in neurons. Nevertheless, the expression of neuronal chemokines and the corresponding chemokine receptors in CNS cells under physiological and pathological conditions indicates that neuronal chemokines contribute to CNS cell interaction. In this study, we review recent studies describing neuronal chemokine expression and discuss potential roles of neuronal chemokines in neuron–astrocyte, neuron–microglia, and neuron–neuron interaction