tram-34 and Glioblastoma

The intermediate-conductance Ca2+-activated K+ channel KCa3.1 is widely expressed in cells of the immune system such as T- and B-lymphocytes, mast cells, macrophages and microglia, but also found in dedifferentiated vascular smooth muscle cells, fibroblasts and many cancer cells including pancreatic, prostate, leukemia and glioblastoma. In all these cell types KCa3.1 plays an important role in cellular activation, migration and proliferation by regulating membrane potential and Ca2+ signaling.. KCa3.1 therefore constitutes an attractive therapeutic target for diseases involving excessive proliferation or activation of one more of these cell types and researchers both in academia and in the pharmaceutical industry have developed several potent and selective small molecule inhibitors of KCa3.1. This article will briefly review the available compounds (TRAM-34, senicapoc, NS6180), their binding sites and mechanisms of action, and then discuss the potential usefulness of these compounds for the treatment of brain tumors based on their brain penetration and their efficacy in reducing microglia activation in animal models of ischemic stroke and Alzheimer's disease.. Senicapoc, which has previously been in Phase III clinical trials, would be available for repurposing, and could be used to quickly translate findings made with other KCa3.1 blocking tool compounds into clinical trials.

Topics: Acetamides; Animals; Brain Neoplasms; Glioblastoma; Humans; Intermediate-Conductance Calcium-Activated Potassium Channels; Membrane Transport Modulators; Pyrazoles; Trityl Compounds

Ca(2+)-activated K(+) channels, such as BK and IK channels, have been proposed to fulfill pivotal functions in neoplastic transformation, malignant progression, and brain infiltration of glioblastoma cells. Here, the ionizing radiation (IR) effect of IK K(+) channel targeting was tested in human glioblastoma cells. IK channels were inhibited pharmacologically by TRAM-34 or genetically by knockdown, cells were irradiated with 6 MV photons and IK channel activity, Ca(2+) signaling, cell cycling, residual double-strand breaks, and clonogenic survival were determined. In addition, the radiosensitizing effect of TRAM-34 was analyzed in vivo in ectopic tumors. Moreover, The Cancer Genome Atlas (TCGA) was queried to expose the dependence of IK mRNA abundance on overall survival (OS) of patients with glioma. Results indicate that radiation increased the activity of IK channels, modified Ca(2+) signaling, and induced a G2-M cell-cycle arrest. TRAM-34 decreased the IR-induced accumulation in G2-M arrest and increased the number of γH2AX foci post-IR, suggesting that TRAM-34 mediated an increase of residual DNA double-strand breaks. Mechanistically, IK knockdown abolished the TRAM-34 effects indicating the IK specificity of TRAM-34. Finally, TRAM-34 radiosensitized ectopic glioblastoma in vivo and high IK mRNA abundance associated with shorter patient OS in low-grade glioma and glioblastoma.. Together, these data support a cell-cycle regulatory function for IK K(+) channels, and combined therapy using IK channel targeting and radiation is a new strategy for anti-glioblastoma therapy.

Topics: Animals; Calcium; Cell Line, Tumor; Combined Modality Therapy; Dacarbazine; Disease-Free Survival; Female; G2 Phase Cell Cycle Checkpoints; Gene Knockdown Techniques; Glioblastoma; Glioma; Histones; Humans; Intermediate-Conductance Calcium-Activated Potassium Channels; Mice; Potassium Channel Blockers; Pyrazoles; Radiation-Sensitizing Agents; RNA, Messenger; Signal Transduction; Temozolomide

Glioblastoma multiforme (GBM) is a diffuse brain tumor characterized by high infiltration in the brain parenchyma rendering the tumor difficult to eradicate by neurosurgery. Efforts to identify molecular targets involved in the invasive behavior of GBM suggested ion channel inhibition as a promising therapeutic approach. To determine if the Ca(2+)-dependent K(+) channel KCa3.1 could represent a key element for GBM brain infiltration, human GL-15 cells were xenografted into the brain of SCID mice that were then treated with the specific KCa3.1 blocker TRAM-34 (1-((2-chlorophenyl) (diphenyl)methyl)-1H-pyrazole). After 5 weeks of treatment, immunofluorescence analyses of cerebral slices revealed reduced tumor infiltration and astrogliosis surrounding the tumor, compared with untreated mice. Significant reduction of tumor infiltration was also observed in the brain of mice transplanted with KCa3.1-silenced GL-15 cells, indicating a direct effect of TRAM-34 on GBM-expressed KCa3.1 channels. As KCa3.1 channels are also expressed on microglia, we investigated the effects of TRAM-34 on microglia activation in GL-15 transplanted mice and found a reduction of CD68 staining in treated mice. Similar results were observed in vitro where TRAM-34 reduced both phagocytosis and chemotactic activity of primary microglia exposed to GBM-conditioned medium. Taken together, these results indicate that KCa3.1 activity has an important role in GBM invasiveness in vivo and that its inhibition directly affects glioma cell migration and reduces astrocytosis and microglia activation in response to tumor-released factors. KCa3.1 channel inhibition therefore constitutes a potential novel therapeutic approach to reduce GBM spreading into the surrounding tissue.

Topics: Animals; Astrocytes; Brain; Brain Neoplasms; Cell Line, Tumor; Cell Movement; Gene Silencing; Glioblastoma; Humans; Intermediate-Conductance Calcium-Activated Potassium Channels; Male; Mice; Mice, Inbred C57BL; Mice, SCID; Neoplasm Invasiveness; Neuroglia; Potassium Channel Blockers; Pyrazoles; RNA, Small Interfering; Xenograft Model Antitumor Assays

In the present study we evaluated the expression of the intermediate conductance calcium-activated potassium (KCa3.1) channel in human glioblastoma stem-like cells (CSCs) and investigated its role in cell motility. While the KCa3.1 channel is not expressed in neuronal- and glial-derived tissues of healthy individuals, both the KCa3.1 mRNA and protein are present in the glioblastoma tumor population, and are significantly enhanced in CSCs derived from both established cell line U87MG and a primary cell line, FCN9. Consistent with these data, voltage-independent and TRAM-34 sensitive potassium currents imputable to the KCa3.1 channel were recorded in the murine GL261 cell line and several primary human glioblastoma cells lines. Moreover, a significantly higher KCa3.1 current was recorded in U87MG-CD133 positive cells as compared to the U87MG-CD133 negative subpopulation. Further, we found that the tumor cell motility is strongly associated with KCa3.1 channel expression. Blockade of the KCa3.1 channel with the specific inhibitor TRAM-34 has in fact a greater impact on the motility of CSCs (reduction of 75%), which express a high level of KCa3.1 channel, than on the FCN9 parental population (reduction of 32%), where the KCa3.1 channel is expressed at lower level. Similar results were also observed with the CSCs derived from U87MG. Because invasion of surrounding tissues is one of the main causes of treatment failure in glioblastoma, these findings can be relevant for future development of novel cancer therapeutic drugs.

Topics: Animals; Cell Line, Tumor; Cell Movement; Fluorescent Antibody Technique; Glioblastoma; Humans; Intermediate-Conductance Calcium-Activated Potassium Channels; Mice; Neoplastic Stem Cells; Patch-Clamp Techniques; Pyrazoles; Real-Time Polymerase Chain Reaction

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.

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Brain Neoplasms; Cell Line, Tumor; Cell Movement; Chloride Channels; Chlorides; Dose-Response Relationship, Drug; Glioblastoma; Humans; Intermediate-Conductance Calcium-Activated Potassium Channels; Membrane Potentials; Neoplasm Invasiveness; Patch-Clamp Techniques; Potassium; Pyrazoles; Serum; Time Factors