Bisacodyl and its cytotoxic activity on human glioblastoma stem-like cells. Implication of inositol 1,4,5-triphosphate receptor dependent calcium signaling.

Glioblastoma is the most common malignant brain tumor. The heterogeneity at the cellular level, metabolic specificities and plasticity of the cancer cells are a challenge for glioblastoma treatment. Identification of cancer cells endowed with stem properties and able to propagate the tumor in animal xenografts has opened a new paradigm in cancer therapy. Thus, to increase efficacy and avoid tumor recurrence, therapies need to target not only the differentiated cells of the tumor mass, but also the cancer stem-like cells. These therapies need to be effective on cells present in the hypoxic, slightly acidic microenvironment found within tumors. Such a microenvironment is known to favor more aggressive undifferentiated phenotypes and a slow-growing "quiescent state" that preserves the cells from chemotherapeutic agents, which mostly target proliferating cells. Based on these considerations, we performed a differential screening of the Prestwick Chemical Library of approved drugs on both proliferating and quiescent glioblastoma stem-like cells and identified bisacodyl as a cytotoxic agent with selectivity for quiescent glioblastoma stem-like cells. In the present study we further characterize bisacodyl activity and show its efficacy in vitro on clonal macro-tumorospheres, as well as in vivo in glioblastoma mouse models. Our work further suggests that bisacodyl acts through inhibition of Ca2+ release from the InsP3 receptors.

Calcium signaling orchestrates glioblastoma development: Facts and conjunctures.

While it is a relatively rare disease, glioblastoma multiform (GBM) is one of the more deadly adult cancers. Following current interventions, the tumor is never eliminated whatever the treatment performed; whether it is radiotherapy, chemotherapy, or surgery. One hypothesis to explain this poor outcome is the "cancer stem cell" hypothesis. This concept proposes that a minority of cells within the tumor mass share many of the properties of adult neural stem cells and it is these that are responsible for the growth of the tumor and its resistance to existing therapies. Accumulating evidence suggests that Ca(2+) might also be an important positive regulator of tumorigenesis in GBM, in processes involving quiescence, maintenance, proliferation, or migration. Glioblastoma tumors are generally thought to develop by co-opting pathways that are involved in the formation of an organ. We propose that the cells initiating the tumor, and subsequently the cells of the tumor mass, must hijack the different checkpoints that evolution has selected in order to prevent the pathological development of an organ. In this article, two main points are discussed. (i) The first is the establishment of a so-called "cellular society," which is required to create a favorable microenvironment. (ii) The second is that GBM can be considered to be an organism, which fights to survive and develop. Since GBM evolves in a limited space, its only chance of development is to overcome the evolutionary checkpoints. For example, the deregulation of the normal Ca(2+) signaling elements contributes to the progression of the disease. Thus, by manipulating the Ca(2+) signaling, the GBM cells might not be killed, but might be reprogrammed toward a new fate that is either easy to cure or that has no aberrant functioning. This article is part of a Special Issue entitled: Calcium and Cell Fate. Guest Editors: Jacques Haiech, Claus Heizmann, Joachim Krebs, Thierry Capiod and Olivier Mignen.

A general framework improving teaching ligand binding to a macromolecule.

The interaction of a ligand with a macromolecule has been modeled following different theories. The tenants of the induced fit model consider that upon ligand binding, the protein-ligand complex undergoes a conformational change. In contrast, the allosteric model assumes that only one among different coexisting conformers of a given protein is suitable to bind the ligand optimally. In the present paper, we propose a general framework to model the binding of ligands to a macromolecule. Such framework built on the binding polynomial allows opening new ways to teach in a unified manner ligand binding, enzymology and receptor binding in pharmacology. Moreover, we have developed simple software that allows building the binding polynomial from the schematic description of the biological system under study. Taking calmodulin as a canonical example, we show here that the proposed tool allows the easy retrieval of previously experimental and computational reports. This article is part of a Special Issue entitled: Calcium Signaling in Health and Disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.

A general framework to characterize inhibitors of calmodulin: use of calmodulin inhibitors to study the interaction between calmodulin and its calmodulin binding domains.

The prominent role of Ca(2+) in cell physiology is mediated by a whole set of proteins involved in Ca(2+)-signal generation, deciphering and arrest. Among these intracellular proteins, calmodulin (CaM) known as a prototypical calcium sensor, serves as a ubiquitous carrier of the intracellular calcium signal in all eukaryotic cell types. CaM is assumed to be involved in many diseases including Parkinson, Alzheimer, and rheumatoid arthritis. Defects in some of many reaction partners of CaM might be responsible for disease symptoms. Several classes of drugs bind to CaM with unwanted side effects rather than specific therapeutic use. Thus, it may be more promising to concentrate at searching for pharmacological interferences with the CaM target proteins, in order to find tools for dissecting and investigating CaM-regulatory and modulatory functions in cells. In the present study, we have established a screening assay based on fluorescence polarization (FP) to identify a diverse set of small molecules that disrupt the regulatory function of CaM. The FP-based CaM assay consists in the competition of two fluorescent probes and a library of chemical compounds for binding to CaM. Screening of about 5300 compounds (Strasbourg Academic Library) by displacement of the probe yielded 39 compounds in a first step, from which 6 were selected. Those 6 compounds were characterized by means of calorimetry studies and by competitive displacement of two fluorescent probes interacting with CaM. Moreover, those small molecules were tested for their capability to displace 8 different CaM binding domains from CaM. Our results show that these CaM/small molecules interactions are not functionally equivalent. The strategy that has been set up for CaM is a general model for the development and validation of other CaM interactors, to decipher their mode of action, or rationally design more specific CaM antagonists. Moreover, this strategy may be used for other protein binding assays intended to screen for molecules with preferred binding activity. This article is part of a Special Issue entitled: 12th European Symposium on Calcium.

A general strategy to characterize calmodulin-calcium complexes involved in CaM-target recognition: DAPK and EGFR calmodulin binding domains interact with different calmodulin-calcium complexes.

Calmodulin (CaM) is a ubiquitous Ca(2+) sensor regulating many biochemical processes in eukaryotic cells. Its interaction with a great variety of different target proteins has led to the fundamental question of its mechanism of action. CaM exhibits four "EF hand" type Ca(2+) binding sites. One way to explain CaM functioning is to consider that the protein interacts differently with its target proteins depending on the number of Ca(2+) ions bound to it. To test this hypothesis, the binding properties of three entities known to interact with CaM (a fluorescent probe and two peptide analogs to the CaM binding sites of death associated protein kinase (DAPK) and of EGFR) were investigated using a quantitative approach based on fluorescence polarization (FP). Probe and peptide interactions with CaM were studied using a titration matrix in which both CaM and calcium concentrations were varied. Experiments were performed with SynCaM, a hybrid CaM able to activate CaM dependent enzymes from mammalian and plant cells. Results show that the interaction between CaM and its targets is regulated by the number of calcium ions bound to the protein, namely one for the DAPK peptide, two for the probe and four for the EGFR peptide. The approach used provides a new tool to elaborate a typology of CaM-targets, based on their recognition by the various CaM-Ca(n) (n=0-4) complexes. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.

[System biology and synthetic biology modify drug discovery and development]. / Biologie des systèmes et ingénierie biologique modifient la découverte et le développement des médicaments.

Life Sciences are built on observations. Right now, a more systemic approach allowing to integrate the different organizational levels in Biology is emerging. Such an approach uses a set of technologies and strategies allowing to build models that appear to be more and more predictive (omics, bioinformatics, integrative biology, computational biology…). Those models accelerate the rational development of new therapies avoiding an engineering based only on trials and errors. This approach both holistic and predictive radically modifies the discovery and development modalities used today in health industries. Moreover, because of the apparition of new jobs at the interface of disciplines, of private and public sectors and of life sciences and engineering sciences, this implies to rethink the training programs in both their contents and their pedagogical tools.

Personalized medicine and education: the challenge.

Although personalized medicine appears to be a truism, medical doctors are still generally trained in an old-fashioned manner with a focus on reactive treatment. The aim of this paper is to emphasize the evolution of life sciences into a more predictive science, where the development of quantitative models is starting to take place. Personalized medicine is a consequence of such paradigm shift. To keep up with the change, the various actors within the health system must be trained in a completely different manner, focusing on the ability to work as part of a multidisciplinary team that includes medical doctors, nurses, engineers in medical imaging, and others who collect information from patients. In addition, these teams should include modelers that are able to integrate the flood of data into predictive and quantitative models. The challenge of implementing new training methods in line with the shift is a major bottleneck to the emergence and success of personalized medicine in our societies.

Calcium fingerprints induced by calmodulin interactors in eukaryotic cells.

Calcium (Ca2+) is a ubiquitous second messenger which promotes cell responses through transient changes in intracellular concentrations. The prominent role of Ca2+ in cell physiology is mediated by a whole set of proteins constituting a Ca2+-signalling toolkit involved in Ca2+-signal generation, deciphering and arrest. The different Ca2+-signalosomes deliver Ca2+-signals with spatial and temporal dynamics to control the function of specific cell types. Among the intracellular proteins involved in Ca2+-signal deciphering, calmodulin (CaM) plays a pivotal role in controlling Ca2+-homeostasis and downstream Ca2+-based signalling events. Due to its ubiquitous expression in eukaryotic cells and the variety of proteins it interacts with, CaM is central in Ca2+-signalling networks. For these reasons, it is expected that disrupting or modifying CaM interactions with its target proteins will affect Ca2+-homeostasis and cellular responses. The resulting calcium response will vary depending on which interactions between CaM and target proteins are altered by the molecules and on the specific Ca2+-toolkit expressed in a given cell, even in the resting state. In the present paper, the effect of six classical CaM interactors (W5, W7, W12, W13, bifonazole and calmidazolium) was studied on Ca2+-signalling in tumor initiating cells isolated from human glioblastoma (TG1) and tobacco cells (BY-2) using the fluorescent Ca2+-sensitive Indo-1 dye and aequorin, respectively. Various Ca2+-fingerprints were obtained depending both on the CaM interactor used and the cell type investigated. These data demonstrate that interaction between the antagonists and CaM results in a differential inhibition of CaM-dependent proteins involved in Ca2+-signal regulation. In addition, the distinct Ca2+-fingerprints in tobacco and human tumor initiating glioblastoma cells induced by a given CaM interactor highlight the specificity of the Ca2+-signalosome in eukaryotic cells.

Using Data Mining to Explore Calmodulin Bibliography.

In this chapter, we present a strategy and the techniques to approach a scientific field from a set of articles gathered from the bibliographic database "Web of Science." The strategy is based on methods developed to analyze social networks. We illustrate its use in studying the calmodulin field. The method allows to structure a huge number of articles when writing a review, to detect the key opinion leaders in a given field, and to locate their own research topic in the landscape of themes deciphered by our own community.We show that the free software VOSviewer may be used without knowledge in computing science and with a short learning period.

Facts and conjectures on calmodulin and its cousin proteins, parvalbumin and troponin C.

This review aims at giving a rational frame to understand the diversity of EF hand containing calcium binding proteins and their roles, with special focus on three members of this huge protein family, namely calmodulin, troponin C and parvalbumin. We propose that these proteins are members of structured macromolecular complexes, termed calcisomes, which constitute building devices allowing treatment of information within eukaryotic cells and namely calcium signals encoding and decoding, as well as control of cytosolic calcium levels in resting cells. Calmodulin is ubiquitous, present in all eukaryotic cells, and pleiotropic. This may be explained by its prominent role in regulating calcium movement in and out of the cell, thus maintaining calcium homeostasis which is fundamental for cell survival. The protein is further involved in decoding transient calcium signals associated with calcium movements after cell stimulation. We will show that the specificity of calmodulin's actions may be more easily explained if one considers its role in the light of calcisomes. Parvalbumin should not be considered as a simple intracellular calcium buffer. It is also a key factor for regulating calcium homeostasis in specific cells that need a rapid retrocontrol of calcium transients, such as fast muscle fibers. Finally, we propose that troponin C, with its four calcium binding domains distributed between two lobes presenting different calcium binding kinetics, exhibits all the characteristics needed to trigger and then post modulate muscle contraction and thus appears as a typical Feed Forward Loop system. If the present conjectures prove accurate, the way will be paved for a new pharmacology targeting the cell calcium signaling machinery. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.

Lebbeckoside C, a new triterpenoid saponin from the stem barks of Albizia lebbeck inhibits the growth of human glioblastoma cells.

One new acacic acid-type saponin, named lebbeckoside C (1), was isolated from the stem barks of Albizia lebbeck. Its structure was established on the basis of extensive analysis of 1D and 2D NMR (1H, 13C NMR, DEPT, COSY, TOCSY, ROESY, HSQC and HMBC) experiments, HRESIMS studies, and by chemical evidence as 3-O-[ß-d-xylopyranosyl-(l→2)-ß-d-fucopyranosyl-(1→6)-[ß-d-glucopyranosyl(1→2)]-ß-d-glucopyranosyl]-21-O-{(2E,6S)-6-O-{4-O-[(2E,6S)-2,6-dimethyl-6-O-(ß-d-quinovopyranosyl)octa-2,7-dienoyl]-4-O-[(2E,6S)-2,6-dimethyl-6-O-(ß-d-quinovopyranosyl)octa-2,7-dienoyl]-ß-d-quinovopyranosyl}-2,6-dimethylocta-2,7-dienoyl}acacic acid 28 O-[ß-d-quinovopyranosyl-(l→3)-[α-l-arabinofuranosyl-(l→4)]-α-l-rhamnopyranosyl-(l→2)-ß-d-glucopyranosyl] ester. The isolated saponin (1) displayed significant cytotoxic activity against the human glioblastoma cell line U-87 MG and TG1 stem-like glioma cells isolated from a patient tumor with IC50 values of 1.69 and 1.44 µM, respectively.

Quiescence status of glioblastoma stem-like cells involves remodelling of Ca2+ signalling and mitochondrial shape.

Quiescence is a reversible cell-cycle arrest which allows cancer stem-like cells to evade killing following therapies. Here, we show that proliferating glioblastoma stem-like cells (GSLCs) can be induced and maintained in a quiescent state by lowering the extracellular pH. Through RNAseq analysis we identified Ca2+ signalling genes differentially expressed between proliferating and quiescent GSLCs. Using the bioluminescent Ca2+ reporter EGFP-aequorin we observed that the changes in Ca2+ homeostasis occurring during the switch from proliferation to quiescence are controlled through store-operated channels (SOC) since inhibition of SOC drives proliferating GSLCs to quiescence. We showed that this switch is characterized by an increased capacity of GSLCs' mitochondria to capture Ca2+ and by a dramatic and reversible change of mitochondrial morphology from a tubular to a donut shape. Our data suggest that the remodelling of the Ca2+ homeostasis and the reshaping of mitochondria might favours quiescent GSLCs' survival and their aggressiveness in glioblastoma.

Ca2+-Dependent Transcriptional Repressors KCNIP and Regulation of Prognosis Genes in Glioblastoma.

Glioblastomas (GBMs) are the most aggressive and lethal primary astrocytic tumors in adults, with very poor prognosis. Recurrence in GBM is attributed to glioblastoma stem-like cells (GSLCs). The behavior of the tumor, including proliferation, progression, invasion, and significant resistance to therapies, is a consequence of the self-renewing properties of the GSLCs, and their high resistance to chemotherapies have been attributed to their capacity to enter quiescence. Thus, targeting GSLCs may constitute one of the possible therapeutic challenges to significantly improve anti-cancer treatment regimens for GBM. Ca2+ signaling is an important regulator of tumorigenesis in GBM, and the transition from proliferation to quiescence involves the modification of the kinetics of Ca2+ influx through store-operated channels due to an increased capacity of the mitochondria of quiescent GSLC to capture Ca2+. Therefore, the identification of new therapeutic targets requires the analysis of the calcium-regulated elements at transcriptional levels. In this review, we focus onto the direct regulation of gene expression by KCNIP proteins (KCNIP1-4). These proteins constitute the class E of Ca2+ sensor family with four EF-hand Ca2+-binding motifs and control gene transcription directly by binding, via a Ca2+-dependent mechanism, to specific DNA sites on target genes, called downstream regulatory element (DRE). The presence of putative DRE sites on genes associated with unfavorable outcome for GBM patients suggests that KCNIP proteins may contribute to the alteration of the expression of these prognosis genes. Indeed, in GBM, KCNIP2 expression appears to be significantly linked to the overall survival of patients. In this review, we summarize the current knowledge regarding the quiescent GSLCs with respect to Ca2+ signaling and discuss how Ca2+ via KCNIP proteins may affect prognosis genes expression in GBM. This original mechanism may constitute the basis of the development of new therapeutic strategies.

WNK1 kinase and its partners Akt, SGK1 and NBC-family Na+/HCO3- cotransporters are potential therapeutic targets for glioblastoma stem-like cells linked to Bisacodyl signaling.

Glioblastoma is a highly heterogeneous brain tumor. The presence of cancer cells with stem-like and tumor initiation/propagation properties contributes to poor prognosis. Glioblastoma cancer stem-like cells (GSC) reside in hypoxic and acidic niches favoring cell quiescence and drug resistance. A high throughput screening recently identified the laxative Bisacodyl as a cytotoxic compound targeting quiescent GSC placed in acidic microenvironments. Bisacodyl activity requires its hydrolysis into DDPM, its pharmacologically active derivative. Bisacodyl was further shown to induce tumor shrinking and increase survival in in vivo glioblastoma models. Here we explored the cellular mechanism underlying Bisacodyl cytotoxic effects using quiescent GSC in an acidic microenvironment and GSC-derived 3D macro-spheres. These spheres mimic many aspects of glioblastoma tumors in vivo, including hypoxic/acidic areas containing quiescent cells. Phosphokinase protein arrays combined with pharmacological and genetic modulation of signaling pathways point to the WNK1 serine/threonine protein kinase as a mediator of Bisacodyl cytotoxic effect in both cell models. WNK1 partners including the Akt and SGK1 protein kinases and NBC-family Na+/HCO3- cotransporters were shown to participate in the compound's effect on GSC. Overall, our findings uncover novel potential therapeutic targets for combatting glioblastoma which is presently an incurable disease.

Use of a fluorescent polarization based high throughput assay to identify new calmodulin ligands.

In order to develop a fluorescence polarization (FP) assay for calcium binding proteins, a fluorescent peptides based library of 1328 compounds has been synthesized. The use of this library has been validated by setting up a FP-high-throughput screening (FP-HTS) assay for calmodulin using the synthetic gene product (synCaM). With this assay, a set of 880 FDA approved compounds was screened. Besides the promazine class, we discovered two new classes of compounds that interact with calmodulin in a calcium dependent manner. One class has compounds with anti-histaminic/spasmolytic activities, and the other one are detergents with antibacterial activities.

Cancer Stem Cell Quiescence and Plasticity as Major Challenges in Cancer Therapy.

Cells with stem-like properties, tumorigenic potential, and treatment-resistant phenotypes have been identified in many human malignancies. Based on the properties they share with nonneoplastic stem cells or their ability to initiate and propagate tumors in vivo, such cells were designated as cancer stem (stem-like) or tumor initiating/propagating cells. Owing to their implication in treatment resistance, cancer stem cells (CSCs) have been the subject of intense investigation in past years. Comprehension of CSCs' intrinsic properties and mechanisms they develop to survive and even enhance their aggressive phenotype within the hostile conditions of the tumor microenvironment has reoriented therapeutic strategies to fight cancer. This report provides selected examples of malignancies in which the presence of CSCs has been evidenced and briefly discusses methods to identify, isolate, and functionally characterize the CSC subpopulation of cancer cells. Relevant biological targets in CSCs, their link to treatment resistance, proposed targeting strategies, and limitations of these approaches are presented. Two major aspects of CSC physiopathology, namely, relative in vivo quiescence and plasticity in response to microenvironmental cues or treatment, are highlighted. Implications of these findings in the context of the development of new therapies are discussed.

The anti-hypertensive drug prazosin inhibits glioblastoma growth via the PKCδ-dependent inhibition of the AKT pathway.

A variety of drugs targeting monoamine receptors are routinely used in human pharmacology. We assessed the effect of these drugs on the viability of tumor-initiating cells isolated from patients with glioblastoma. Among the drugs targeting monoamine receptors, we identified prazosin, an α1- and α2B-adrenergic receptor antagonist, as the most potent inducer of patient-derived glioblastoma-initiating cell death. Prazosin triggered apoptosis of glioblastoma-initiating cells and of their differentiated progeny, inhibited glioblastoma growth in orthotopic xenografts of patient-derived glioblastoma-initiating cells, and increased survival of glioblastoma-bearing mice. We found that prazosin acted in glioblastoma-initiating cells independently from adrenergic receptors. Its off-target activity occurred via a PKCδ-dependent inhibition of the AKT pathway, which resulted in caspase-3 activation. Blockade of PKCδ activation prevented all molecular changes observed in prazosin-treated glioblastoma-initiating cells, as well as prazosin-induced apoptosis. Based on these data, we conclude that prazosin, an FDA-approved drug for the control of hypertension, inhibits glioblastoma growth through a PKCδ-dependent mechanism. These findings open up promising prospects for the use of prazosin as an adjuvant therapy for glioblastoma patients.

The EF-Handome: combining comparative genomic study using FamDBtool, a new bioinformatics tool, and the network of expertise of the European Calcium Society.

By combining a bioinformatics tool (FamDBTool) and the expertise of a network of calcium binding proteins specialists (European Calcium Society), we aim to accelerate and to rationalize the curation of the public general biological database such as Swissprot and Entrez Gene with respect to specific protein families. In this paper, we show the feasibility of such rationale in order to set and to curate the human, mouse and rat sets of EF-Hand genes, the EF-Handome.

Glioblastoma and calcium signaling--analysis of calcium toolbox expression.

The characteristics of a cellular calcium signal (calcium signature) are determined, at least partly, by the expression of a subset of genes encoding proteins involved in calcium entry, calcium uptake and calcium modulation. Our aim in the present work was to characterize the set of genes involved in calcium signal generation that are differentially expressed in normal brain tissues versus brain tumor and/or glioma stem cells. Public datasets were analyzed according to a four step methodology consisting of: 1. detecting the outliers by using principal component analysis of the whole transcriptome; 2. building a calcium toolbox composed of 260 genes involved in the generation and modulation of the calcium signal; 3. analyzing the calcium toolbox transcriptome of different human brain areas and 4. detecting genes from the calcium toolbox preferentially expressed in tumor tissues or tumor cells compared to normal brain tissues. Our approach was validated on normal brain tissue. Tumor sample analysis allowed us to disclose a set of eighteen genes characteristic of glioblastoma tissues or glioma stem cells. Interpreting the set of genes highlighted in the study led us to propose that i) the mechanism of store operated calcium entry is strongly perturbed in cancer cells and tissues, ii) the process of calcium reuptake into mitochondria is more important in cancer cells and tissues than in their normal counterparts and iii) these two mechanisms may be coupled in at least one subgroup of the glioblastoma stem cells.

Triterpenoid saponins from Albizia lebbeck (L.) Benth and their inhibitory effect on the survival of high grade human brain tumor cells.

As part of our search of new bioactive triterpenoid saponins from Cameroonian Mimosaceae plants, phytochemical investigation of the roots of Albizia lebbeck led to the isolation of two new oleanane-type saponins, named lebbeckosides A-B (1-2). Their structures were established on the basis of extensive 1D and 2D NMR ((1)H, (13)C NMR, DEPT, COSY, TOCSY, ROESY, HSQC, and HMBC) and HRESIMS studies, and by chemical evidence. Compounds 1-2 were evaluated for their inhibitory effect on the metabolism of high grade human brain tumor cells, the human glioblastoma U-87 MG cell lines and the glioblastoma stem-like TG1 cells isolated from a patient tumor, and known to be particularly resistant to standard therapies. The isolated saponins showed significant cytotoxic activity against U-87 MG and TG1 cancer cells with IC50 values of 3.46 µM and 1.36 µM for 1, and 2.10 µM and 2.24 µM for 2, respectively.