Bernard Fioretti

CXCL12-induced glioblastoma cell migration requires intermediate conductance Ca2+-activated K+ channel activity

The activation of ion channels is crucial during cell movement, including glioblastoma cell invasion in the brain parenchyma. In this context, we describe for the first time the contribution of intermediate conductance Ca(2+)-activated K (IK(Ca)) channel activity in the chemotactic response of human glioblastoma cell lines, primary cultures, and freshly dissociated tissues to CXC chemokine ligand 12 (CXCL12), a chemokine whose expression in glioblastoma has been correlated with its invasive capacity. We show that blockade of the IK(Ca) channel with its specific inhibitor 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34) or IK(Ca) channel silencing by short hairpin RNA (shRNA) completely abolished CXCL12-induced cell migration. We further demonstrate that this is not a general mechanism in glioblastoma cell migration since epidermal growth factor (EGF), which also activates IK(Ca) channels in the glioblastoma-derived cell line GL15, stimulate cell chemotaxis even if the IK(Ca) channels have been blocked or silenced. Furthermore, we demonstrate that both CXCL12 and EGF induce Ca(2+) mobilization and IK(Ca) channel activation but only CXCL12 induces a long-term upregulation of the IK(Ca) channel activity. Furthermore, the Ca(2+)-chelating agent BAPTA-AM abolished the CXCL12-induced, but not the EGF-induced, glioblastoma cell chemotaxis. In addition, we demonstrate that the extracellular signal-regulated kinase (ERK)1/2 pathway is only partially implicated in the modulation of CXCL12-induced glioblastoma cell movement, whereas the phosphoinositol-3 kinase (PI3K) pathway is not involved. In contrast, EGF-induced glioblastoma migration requires both ERK1/2 and PI3K activity. All together these findings suggest that the efficacy of glioblastoma invasiveness might be related to an array of nonoverlapping mechanisms activated by different chemotactic agents.

KCa3.1 channels are involved in the infiltrative behavior of glioblastoma in vivo

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 Ca2+-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.

Intermediate conductance Ca2+-activated potassium channel (KCa3.1) in the inner mitochondrial membrane of human colon cancer cells

Patch-clamping mitoplasts isolated from human colon carcinoma 116 cells has allowed the identification and characterization of the intermediate conductance Ca(2+)-activated K(+)-selective channel K(Ca)3.1, previously studied only in the plasma membrane of various cell types. Its identity has been established by its biophysical and pharmacological properties. Its localisation in the inner membrane of mitochondria is indicated by Western blots of subcellular fractions, by recording of its activity in mitochondria made fluorescent by a mitochondria-targeted fluorescent protein and by the co-presence of channels considered to be markers of the inner membrane. Moderate increases of mitochondrial matrix [Ca(2+)] will cause mtK(Ca)3.1 opening, thus linking inner membrane K(+) permeability and transmembrane potential to Ca(2+) signalling.

Expression and Modulation of the Intermediate- Conductance Ca2+-Activated K+ Channel in Glioblastoma GL-15 Cells

We report here the expression and properties of the intermediate-conductance Ca2+-activated K+ (IKCa) channel in the GL-15 human glioblastoma cell line. Macroscopic IKCa currents on GL-15 cells displayed a mean amplitude of 7.2±0.8 pA/pF at 0 mV, at day 1 after plating. The current was inhibited by clotrimazole (CTL, IC50=257 nM), TRAM-34 (IC50=55 nM), and charybdotoxin (CTX, IC50=10.3 nM). RT-PCR analysis demonstrated the expression of mRNA encoding the IKCa channel in GL-15 cells. Unitary currents recorded using the inside-out configuration had a conductance of 25 pS, a KD for Ca2+ of 188 nM at -100 mV, and no voltage dependence. We tested whether the IKCa channel expression in GL-15 cells could be the result of an increased ERK activity. Inhibition of the ERK pathway with the MEK antagonist PD98059 (25 µM, for 5 days) virtually suppressed the IKCa current in GL-15 cells. PD98059 treatment also increased the length of cellular processes and up-regulated the astrocytic differentiative marker GFAP. A significant reduction of the IKCa current amplitude was also observed with time in culture, with mean currents of 7.17±0.75 pA/pF at 1-2 days, and 3.11±1.35 pA/pF at 5-6 days after plating. This time-dependent downregulation of the IKCa current was not accompanied by changes in the ERK activity, as assessed by immunoblot analysis. Semiquantitative RT-PCR analysis demonstrated a ~35% reduction of the IKCa channel mRNA resulting from ERK inhibition and a ~50% reduction with time in culture.

Serum-activated K and Cl currents underlay U87-MG glioblastoma cell migration

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 (IKCa) 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 IKCa 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. J. Cell. Physiol. 226: 1926–1933, 2011.

The Inhibition of KCa3.1 Channels Activity Reduces Cell Motility in Glioblastoma Derived Cancer Stem Cells

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.

Histamine hyperpolarizes human glioblastoma cells by activating the intermediate-conductance Ca2+-activated K+ channel

The effects of histamine on the membrane potential and currents of human glioblastoma (GL-15) cells were investigated. In perforated whole cell configuration, short (3 s) applications of histamine (100 microM) hyperpolarized the membrane by activating a K(+)-selective current. The response involved the activation of the pyrilamine-sensitive H(1) receptor and Ca(2+) release from thapsigargin-sensitive intracellular stores. The histamine-activated current was insensitive to tetraethylammonium (3 mM), iberiotoxin (100 nM), and d-tubocurarine (100 microM) but was markedly inhibited by charybdotoxin (100 nM), clotrimazole (1 microM), and 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34, 1 microM), a pharmacological profile congruent with the intermediate conductance Ca(2+)-activated K(+) (IK(Ca)) channel. Cell-attached recordings confirmed that histamine activated a K(+) channel with properties congruent with the IK(Ca) channel (voltage independence, 22 pS unitary conductance and slight inward rectification in symmetrical 140 mM K(+)). More prolonged histamine applications (2-3 min) often evoked a sustained IK(Ca) channel activity, which depended on a La(2+) (10 microM)-sensitive Ca(2+) influx. Intracellular Ca(2+) measurements revealed that the sustained IK(Ca) channel activity enhanced the histamine-induced Ca(2+) signal, most likely by a hyperpolarization-induced increase in the driving force for Ca(2+) influx. In virtually all cells examined we also observed the expression of the large conductance Ca(2+)-activated K(+) (BK(Ca)) channel, with a unitary conductance of ca. 230 pS in symmetrical 140 mM K(+), and a Ca(2+) dissociation constant [K(D(Ca))] of ca. 3 microM, at -40 mV. Notably in no instance was the BK(Ca) channel activated by histamine under physiological conditions. The most parsimonious explanation based on the different K(D(Ca)) for the two K(Ca) channels is provided.

Extracellular guanosine-5′-triphosphate modulates myogenesis via intermediate Ca2+-activated K+ currents in C2C12 mouse cells

In this study we investigated the role of extracellular 5′‐guanosine‐triphosphate (GTP) on early phases of skeletal muscle differentiation using the widely used C2C12 mouse cells as a myogenic model. We show that extracellular GTP binding to specific sites activates a metabotropic cascade that leads to a transient intracellular Ca2+ mobilization, consequent activation of the intermediate Ca2+‐activated K+ channels (IKCa), and hyperpolarization of the plasma membrane. We further show that in differentiating C2C12 myoblasts GTP induces a proliferative boost, and increases the number of cells positive for the myosin heavy chain (MyHC) proteins. These effects were shown to be mediated by the IKCa channel‐dependent hyperpolarization, as evidenced by their disappearance when myoblasts were incubated with the IKCa channel inhibitor charybdotoxin. These data give new insights into nucleotide purinergic signalling pathways, and address the role of the GTP‐dependent IKCa channel activation and hyperpolarization in myogenesis.

An investigation of the occurrence and properties of the mitochondrial intermediate-conductance Ca2+-activated K+ channel mtKCa3.1

The mitochondrial intermediate-conductance Ca2+-activated K+ channel mtKCa3.1 has recently been discovered in the HCT116 colon tumor-derived cell line, which expresses relatively high levels of this protein also in the plasma membrane. Electrophysiological recordings revealed that the channel can exhibit different conductance states and kinetic modes, which we tentatively ascribe to post-translational modifications. To verify whether the localization of this channel in mitochondria might be a peculiarity of these cells or a more widespread feature we have checked for the presence of mtKCa3.1 in a few other cell lines using biochemical and electrophysiological approaches. It turned out to be present at least in some of the cells investigated. Functional assays explored the possibility that mtKCa3.1 might be involved in cell proliferation or play a role similar to that of the Shaker-type KV1.3 channel in lymphocytes, which interacts with outer mitochondrial membrane-inserted Bax thereby promoting apoptosis (Szabò, I. et al., Proc. Natl. Acad Sci. USA 105 (2008) 14861-14866). A specific KCa3.1 inhibitor however did not have any detectable effect on cell proliferation or death.

Expression and Role of the Intermediate-Conductance Calcium-Activated Potassium Channel KCa3.1 in Glioblastoma

Glioblastomas are characterized by altered expression of several ion channels that have important consequences in cell functions associated with their aggressiveness, such as cell survival, proliferation, and migration. Data on the altered expression and function of the intermediate-conductance calcium-activated K (KCa3.1) channels in glioblastoma cells have only recently become available. This paper aims to (i) illustrate the main structural, biophysical, pharmacological, and modulatory properties of the KCa3.1 channel, (ii) provide a detailed account of data on the expression of this channel in glioblastoma cells, as compared to normal brain tissue, and (iii) critically discuss its major functional roles. Available data suggest that KCa3.1 channels (i) are highly expressed in glioblastoma cells but only scantly in the normal brain parenchima, (ii) play an important role in the control of glioblastoma cell migration. Altogether, these data suggest KCa3.1 channels as potential candidates for a targeted therapy against this tumor.