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The present work describes the isolation and anticancer activity of Tapinanthus sp. which is a hemi parasitic plant harvested on Combretum glutinosum, the host plant. Phytochemical study afforded a...

Plants play an important role in cancer therapy. They are source of natural molecules which can induce apoptosis in cancer cells by affecting molecular mechanisms implicated in cancer progression. The MAP Kinase/ERK1/2 and PI3K/AKT signaling pathways are two classical signaling pathways implicated in cancer progression and constitute therapeutic targets against cancer. This study aimed to evaluate the effect of euphol on MAP Kinase/ERK1/2 and PI3K/AKT signaling pathways in glioblastoma and prostate cancer cells. Euphol is a tetracyclique triterpene alcohol isolated from Tapinanthus sp. which is a hemi parasitic plant belonging to Loranthaceae family.Plant powder was extracted by maceration and euphol was isolated and described using respectively column chromatography separation on silica gel and spectroscopic data. Cytotoxic effect of euphol was evaluated using XTT assay and its effect on MAP Kinase/ERK1/2 and PI3K/AKT protein expression was investigated by Western immunoblot analysis. Apotosis was analyzed by evaluating caspase-3/7 activity.Our investigations demonstrated that this compound has an important cytotoxic effect on C6 and U87 MG glioblastoma (GBM) cells and PC-3 prostate cancer cells. Furthermore, euphol-induced apoptosis revealed by elevated caspase 3/7 activity, was correlated with a significant inhibition of MAP kinase/Erk 1/2 and PI3K/Akt signaling pathway in glioblastoma U87 MG cells. The reverse effect was observed in C6 glioblastoma cells, where apoptosis was correlated with a long-lasting activation of Erk 1/2. In PC-3 cells, euphol had no or limited effect on Erk 1/2 and Akt activity.These results indicate that euphol induces cell death in glioblastoma and prostate cancer cells and regulates significantly Erk1/2 and Akt activity in glioblastoma cells.

Background: Cancer incidence has been growing in an alarming rate worldwide and new therapeutics are needed, particularly for intractable and chemoresistant cases. We evaluated the cytotoxic effects of Combretum fragrans F. Hoffm (Combretaceae) on glioblastoma (U87MG and C6) and prostate (PC-3) cancer cell lines. Methods: The cytotoxic effect of the methanolic extract of the stem bark of Combretum fragrans was assessed using XTT (2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide) test. Expressions of Akt and ERK1/2 were determined using Western blot technique, while Caspase-3/7 kits were used to evaluate caspase-3/7 activity. Results: C. fragrans extract inhibited the proliferation of U87 (IC50 = 20.13 µg/mL), C6 (IC50 = 12.17 µg/mL), and PC-3 (IC50 = 11.50 µg/mL) cells. Treatment with the extract resulted in lower levels (p < 0.001) of phospho-ERK1/2 and phospho-Akt in U87 cells, and instead, higher levels of phospho-ERK1/2 (p < 0.001) in C6 and PC-3 cells. An increase in caspase-3/7 activity was observed, mainly after 24 hours of treatment, indicating the activation of apoptotic processes. Conclusion: Altogether, these results suggest that C. fragrans have potent anticancer properties. This plant should be further investigated for developing new anticancer drugs.

The present work describes the isolation and anticancer activity of

Clustering via marked point processes and influence space, Is-ClusterMPP, is a new unsupervised clustering algorithm through adaptive MCMC sampling of a marked point processes of interacting balls. The designed Gibbs energy cost function makes use of k-influence space information. It detects clusters of different shapes, sizes and unbalanced local densities. It aims at dealing also with high-dimensional datasets. By using the k-influence space, Is-ClusterMPP solves the problem of local heterogeneity in densities and prevents the impact of the global density in the detection of unbalanced classes. This concept reduces also the input values amount. The curse of dimensionality is handled by using a local subspace clustering principal embedded in a weighted similarity metric. Balls covering data points are constituting a configuration sampled from a marked point process (MPP). Due to the choice of the energy function, they tends to cover neighboring data, which share the same cluster. The statistical model of random balls is sampled through a Monte Carlo Markovian dynamical approach. The energy is balancing different goals. (1) The data driven objective function is provided according to k-influence space. Data in a high-dense region are favored to be covered by a ball. (2) An interaction part in the energy prevents the balls full overlap phenomenon and favors connected groups of balls. The algorithm through Markov dynamics, does converge towards configurations sampled from the MPP model. This algorithm has been applied in real benchmarks through gene expression data set of various sizes. Different experiments have been done to compare Is-ClusterMPP against the most well-known clustering algorithms and its efficiency is claimed.

Microarray technologies become a powerful technique for simultaneously monitoring expression patterns of thousands of genes under different conditions. However, it is important to identify gene groups that manifest similar expression profiles and are activated by similar conditions. ClusterMPP: Clustering by Marked Point Process is a new microarray data clustering algorithm performed in two steps. The first one detects cluster modes representing regions of high density observations in the raw space. Based on the simulation of a proposed Marked Point Process by the well-known Reversible Jump Markov Chain Monte Carlo algorithm, where we consider several movements like birth and death, this algorithm step identifies prototype observations of each cluster. The second step of ClusterMPP is the K nearest neighbors (KNN) assignation that affects the remaining observations to the corresponding clusters. We experiment ClusterMPP on several complex and scalable microarray datasets. The results show the efficiency of ClusterMPP compared to well-known microarray data clustering methods like K-means, Spectral Clustering, and Mean-Shift.

In response to the rapid development of DNA Microarray Technologies, many differentially expressed genes selection algorithms have been developed, and different comparison studies of these algorithms have been done. However, it is not clear how these methods compare with each other, especially when we used different developments tools. Here, we considered three commonly used differentially expressed genes selection approaches, namely: Fold Change, T-test and SAM, using Bioinformatics Matlab Toolbox and R/BioConductor. We used two datasets, issued from the affymetrix technology, to present results of used methods and software's in gene selection process. The results, in terms of sensitivity and specificity, indicate that the behavior of SAM is better compared to Fold Change and T-test using R/BioConductor. While, no practical differences were observed between the three gene selection methods when using Bioinformatics Matlab Toolbox. In face of our result, the ROC curve shows that: on the one hand R/BioConductor using SAM is favored for microarray selection compared to the other methods. And, on the other hand, results of the three studied gene selection methods using Bioinformatics Matlab Toolbox are still comparable for the two datasets used.

We have previously shown that CD36 is a membrane protein that facilitates long chain fatty acid (FA) transport by muscle tissues. We also documented the significant impact of muscle CD36 expression on heart function, skeletal muscle insulin sensitivity as well as on overall metabolism. To identify a comprehensive set of genes that are differentially regulated by CD36 expression in the heart, we used two microarray technologies (Affymetrix and Agilent) to compare gene expression in heart tissues from CD36 KnocK-Out (KO-CD36) versus wild type (WT-CD36) mice. The obtained results using the two technologies were similar with around 35 genes differentially expressed using both technologies. Absence of CD36 led to down-regulation of the expression of three groups of genes involved in pathways of FA metabolism, angiogenesis/apoptosis and structure. These data are consistent with the fact that the CD36 protein binds FA and thrombospondin 1 invoved respectively in lipid metabolism and anti-angiogenic activities. In conclusion, our findings led to validate our data analysis workflow and identify specific pathways, possibly underlying the phenotypic abnormalities in CD36 Knock -Out hearts.

Saturated fatty acids (SFA) have been reported to alter organelle integrity and function in many cell types, including muscle and pancreatic β-cells, adipocytes, hepatocytes and cardiomyocytes. SFA accumulation results in increased amounts of ceramides/sphingolipids and saturated phospholipids (PL). In this study, using a yeast-based model that recapitulates most of the trademarks of SFA-induced lipotoxicity in mammalian cells, we demonstrate that these lipid species act at different levels of the secretory pathway. Ceramides mostly appear to modulate the induction of the unfolded protein response and the transcription of nutrient transporters destined to the cell surface. On the other hand, saturated PL, by altering membrane properties, directly impact vesicular budding at later steps in the secretory pathway, i.e. at the trans-Golgi Network level. They appear to do so by increasing lipid order within intracellular membranes which, in turn, alters the recruitment of loose lipid packing-sensing proteins, required for optimal budding, to nascent vesicles. We propose that this latter general mechanism could account for the well-documented deleterious impacts of fatty acids on the last steps of the secretory pathway in several cell types.

The EGF-family of tyrosine-kinase receptors activates cytoplasmic pathways involved in cell proliferation, migration and differentiation in response to specific extracellular ligands. Beside these canonical pathways, the nuclear localization of the ErbB receptors in primary tumours and cancer cell lines led to investigate their role as transcriptional regulators of cancer genes. The nuclear localization of ErbB3 has been reported in various cancer tissues and cell lines but the nuclear functions and the putative correlation with tumour progression and resistance to therapy remain unclear. We first assessed ErbB3 expression in normal and tumour prostate tissues. The nuclear staining was mainly due to an isoform matching the C-terminus domain of the full length ErbB3185kDa receptor. Nuclear staining was also restricted to cancer cells and was increased in advanced castration-resistant prostate cancer when compared to localized tumours, suggesting it could be involved in the progression of prostate cancer up to the terminal castration-resistant stage. ChIP-on-chip experiments were performed on immortalized and tumour cell lines selected upon characterization of endogenous nuclear expression of an ErbB380kDa isoform. Among the 1840 target promoters identified, 26 were selected before ErbB380kDa-dependent gene expression was evaluated by real-time quantitative RT-PCR, providing evidence that ErbB380kDa exerted transcriptional control on those genes. Some targets are already known to be involved in prostate cancer progression even though no link was previously established with ErbB3 membrane and/or nuclear signalling. Many others, not yet associated with prostate cancer, could provide new therapeutic possibilities for patients expressing ErbB380kDa. Detecting ErbB380kDa could thus constitute a useful marker of prognosis and response to therapy.

EGFR family members are tyrosine kinase transmembrane receptors that, in response to specific extracellular ligands, activate cytoplasmic pathways involved in cell proliferation, migration and differentiation. More recently, a pivotal role for EGF receptors has emerged, through the description of their nuclear localization.We report here the characterization of a nuclear variant of the kinase-defective ErbB3 receptor, ErbB3 80 kDa , spanning the intracytoplasmic domain of the receptor. We assessed the putative transcriptional functions of ErbB3 80 KDa in cancer cells, through the regulation of the proliferative Cyclin D1 gene, an already known target of the ErbB3 cytoplasmic signaling. We demonstrate here that the binding of ErbB3 80 KDa on the promoter activates Cyclin D1 transcription and subsequent protein expression, leading to an increased cell proliferation. This mechanism can be balanced in response to the ectopic expression of the tumor suppressor p14 ARF that physically interacts with ErbB3 100 kDa and sequesters it into nucleoli. Our data also show that ErbB3 80 kDa increases the transcription of proliferative genes even though the cytoplasmic pathways are not activated. This nuclear ErbB3 pathway and the target genes concerned need to be further studied. Indeed, such mechanism could explain the tumor relapse observed in response to treatments aimed at blocking the receptor activation in response to ligand binding.

Transient receptor potential (Trp) proteins form ion channels implicated in the calcium entry observed after stimulation of the phospholipase C pathway. Kyte-Doolittle analysis of the amino acid sequence of Trp proteins identifies seven hydrophobic regions (H1-H7) with potential of forming transmembrane segments. A limited sequence similarity to voltage-gated calcium channel alpha1 subunits lead to the prediction of six transmembrane (TM) segments flanked by intracellular N and C termini and a putative pore region between TM5 and TM6. However, experimental evidence supporting this model is missing. Using human Trp 3 to test Trp topology, we now confirm the intracellular nature of the termini by immunocytochemistry. We also demonstrate presence of a unique glycosylation site in position 418, which defines one extracellular loop between H2 and H3. After removal of this site and insertion of ten separate glycosylation sites, we defined two additional extracellular loops between H4 and H5, and H6 and H7. This demonstrated the existence of six transmembrane segments formed of H2-H7. Thus, the first hydrophobic region of Trp rather than being a transmembrane segment is intracellular and available for protein-protein interactions. A site placed in the center of the putative pore region was glycosylated, suggesting that this region may have been luminal and was reinserted into the membrane at a late stage of channel assembly.

The thyrotropin (TSH) and follicle-stimulating hormone (FSH) receptors are present mainly on the basolateral cell surface in the thyroid gland and in Sertoli cells, whereas in ovarian and in testicular cells, the luteinizing hormone (LH) receptors are distributed throughout the cell surface. When expressed in Madin-Darby canine kidney (MDCK) cells, all three receptors accumulated at the basolateral cell surface showing that they carry the corresponding targeting signals. The receptors were directly delivered to the basolateral surface of the MDCK cells. A minor fraction of the gonadotropin receptors but not of TSH receptors was secondarily targeted to the apical surface through transcytosis. The mechanisms of basolateral targeting and transcytosis were analyzed using the FSH receptor as a model. Both were insensitive to brefeldin A and pertussis toxin. Gs activation by AlF4− and cholera toxin provoked a marked enhancement of FSH receptor transcytosis. The population of Gs proteins involved in this mechanism was different from that involved in signal transduction since neither FSH nor forskolin mimicked the effects of AlF4− and cholera toxin. Gs activation provoked a similar effect on LH receptor distribution in MDCK cells, whereas it did not modify the compartmentalization of the TSH receptor. Hormone-specific transcytosis was observed in MDCK cells expressing the gonadotropin (FSH and LH) receptors and was increased after cholera toxin administration.

Motivation: In the analysis of experiments that involves the high density of oligonucleotide chips, it is important to generate list of genes or ‘targets’ from the genome wide data set that contains a lot of information. Gene selection is a process that seeks to identify the most significant genes which reveal large expressions changes between the baseline experiments and the conditions. Even though, several algorithms like T-test and other derived statistical algorithms were used for that selection process, the suitable Pvalue Cutoff remains difficult to choose. Therefore, one solution consists of using a False Discovery Rate (FDR) control. The Significance Analysis of Microarray (SAM) and the T-test Benjamini & Hochberg (BH) algorithms have been successfully used in such way. However, the reproductivity of results and their impact on the genes and/or experiments classification, while using different soft tools remain a subject of discussion. Methods: we use two Affymetrix data sets, when we look for identifying list of genes under SAM and T-test-BH algorithms with FDR control running under R/Bioconductor project and Bioinformtics Tool Box of Math works and Expander. Results: The list of selected genes changes significantly when using the two algorithms under R/Bioconductor project, Bioinformtics Tool Box of Math works and Expander. By means of data provided from publicly databases, we illustrate, that the permutation process of the multiple statistical T-test (SAM and BH) may affect results of selection process. Moreover, list of genes using the two Soft is affected by the choice of the Pvalue Cutoff for identifying true differential expressed genes. According to this work, we present some results clarifying sensitivity and efficiency of used soft and its influence in gene selection process. Hierarchical classification of selected genes and corresponding experiences confirm the influence of both methods and tools on the outcome of gene expression data analysis.

During the last 2 years, our laboratory has worked on the elucidation of the molecular basis of capacitative calcium entry (CCE) into cells. Specifically, we tested the hypothesis that CCE channels are formed of subunits encoded in genes related to the Drosophila trp gene. The first step in this pursuit was to search for mammalian trp genes. We found not one but six mammalian genes and cloned several of their cDNAs, some in their full length. As assayed in mammalian cells, overexpression of some mammalian Trps increases CCE, while expression of partial trp cDNAs in antisense orientation can interfere with endogenous CCE. These findings provided a firm connection between CCE and mammalian Trps. This article reviews the known forms of CCE and highlights unanswered questions in our understanding of intracellular Ca 2+ homeostasis and the physiological roles of CCE.

The extracellular and intracellular domains of the human thyrotropin receptor were expressed in Escherichia coli and the proteins were used to produce monoclonal anti-receptor antibodies. Immunoblot studies and immunoaffinity purification showed that the receptor is composed of two subunits linked by disulfide bridges and probably derived by proteolytic cleavage of a single 90-kDa precursor. The extracellular alpha subunit (hormone binding) had an apparent molecular mass of 53 kDa (35 kDa after deglycosylation with N-glycosidase F). The membrane-spanning beta subunit seemed heterogeneous and had an apparent molecular mass of 33-42 kDa. Human thyroid membranes contained a 2.5- to 3-fold excess of beta subunits over alpha subunits. Immunocytochemistry showed the presence of both subunits in all the follicular thyroid cells, and both subunits were restricted to the basolateral region of the cell membrane.

Phosphodiesterase 6 (PDE6) is the primary effector of phototransduction in vertebrate photoreceptors. Previous studies described the expression of the regulatory subunit of rod PDE6 (Pgamma-rod) in non-photosensitive tissues of the adult rat and the effects of this protein on MAP kinase pathways. Upon examination of the Pgamma-rod sequence, we detected a proline-rich domain that might reveal its ability to interact with SH3-containing proteins. Therefore, the present study was initiated to identify new protein partners of Pgamma-rod.A yeast two-hybrid screen of a rat brain cDNA library was performed using Pgamma-rod as a bait. Pgamma-rod-SH3 interaction was confirmed by GST pull-down of in vitro-translated proteins. The aminoacids involved in the interaction were mapped by site-directed mutagenesis. Rnase protection assay, RT-PCR and western blot analysis were used to detect Pgamma-rod expression in various rat tissues.A clone was isolated twice, that consisted essentially of the SH3 domain of the formin-binding protein 17 (FBP17). This interaction was confirmed by GST pull-down. Mutational analysis of the Pgamma-rod-FBP17 interaction confirmed it involved the proline-rich domain of Pgamma-rod and the SH3 domain of FBP17. This proline-rich domain also allowed Pgamma-rod to interact with Cdc42-interacting protein 4 (CIP4), another SH3-containing protein. RT-PCR and Rnase protection assay detected different amounts of Pgamma-rod mRNA in adult and embryonic rat tissues. Western blots confirmed the presence of low levels of Pgamma-rod protein only in embryonic tissues.Our data suggest that Pgamma-rod participates in SH3-mediated cellular pathways and may therefore play a wider role than previously appreciated. One possibility is that FBP17 interaction with sorting nexin 2 might connect Pgamma-rod to receptor tyrosine kinase recycling. However, further studies are still required to identify the diversity of SH3-containing proteins that interact with Pgamma-rod. This effort should provide a rationale to understand how Pgamma-rod can affect receptor internalization-dependent MAP kinase activity.

The regulatory mechanisms of oocyte maturation remain poorly understood. Although gonadotropins play a major role in these processes, they have generally been considered to act on somatic supportive cells, but not directly on germ cells. We have raised high affinity monoclonal antibodies against LH and FSH receptors. When using the latter to study receptor distribution in human and pig ovaries we have observed the presence of FSH (but not LH) receptors in the oocytes. FSH receptors appeared in the oocytes of primary follicles during follicular development and persisted up to the preovulatory stage. In denuded human preovulatory oocytes, FSH receptor mRNA was detected at a concentration per cell exceeding by about 20-fold that present in granulosa cells. Saturable binding of [125I]FSH to the membrane of oocytes was demonstrated by autoradiography. When incubated with FSH, denuded oocytes responded by a mobilization of Ca2+. These observations concur to demonstrate the presence of functional FSH receptors in oocytes and raise the possibility of direct control of oocyte development by FSH.

Microarray experiments enable the simultaneous measure of expression levels of large amount of genes and have many applications. A widespread one is finding set of genes that are differentially expressed. Significance Analysis of Microarrays (SAM) helps to produce those sets using multiple testing techniques. There is unfortunately not yet a public tool enabling to do SAM using the Matlab platform. We here define MatSAM, a SAM implementation in Matlab, and show that it yields results of high confidence comparatively to those obtained by putative tools available in the R programming environment. MatSAM can be used in conjunction with Matlab Bioinformatics toolbox to perform further analysis. Availability : MatSAM is available as source code at http://www.bioinfoindia.org/MatSAM

Capacitative Ca 2+ entry (CCE) is Ca 2+ entering after stimulation of inositol 1,4,5-trisphosphate (IP3) formation and initiation of Ca 2+ store depletion. One hallmark of CCE is that it can also be triggered merely by store depletion, as occurs after inhibition of internal Ca 2+ pumps with thapsigargin. Evidence has accumulated in support of a role of transient receptor potential (Trp) proteins as structural subunits of a class of Ca 2+ -permeable cation channels activated by agonists that stimulate IP3 formation—very likely through a direct interaction between the IP3 receptor and a Trp subunit of the Ca 2+ entry channel. The role of Trp’s in Ca 2+ entry triggered by store depletion alone is less clear. Only a few of the cloned Trp’s appear to enhance this type of Ca 2+ entry, and when they do, the effect requires special conditions to be observed, which native CCE does not. Here we report the full-length cDNA of mouse trp2 , the homologue of the human trp2 pseudogene. Mouse Trp2 is shown to be readily activated not only after stimulation with an agonist but also by store depletion in the absence of an agonist. In contrast to other Trp proteins, Trp2-mediated Ca 2+ entry activated by store depletion is seen under the same conditions that reveal endogenous store depletion-activated Ca 2+ entry, i.e., classical CCE. The findings support the general hypothesis that Trp proteins are subunits of store- and receptor-operated Ca 2+ channels.

The follicle-stimulating hormone receptor (FSHR) is physiologically localized in the basolateral compartment of the membrane of Sertoli cells. This localization is also observed when the receptor is experimentally expressed in Madin-Darby canine kidney cells. We thus used in vitro mutagenesis and transfection into these polarized cells to delineate the basolateral localization signal of the receptor. The signal was localized in the C-terminal tail of the intracellular domain (amino acids 678–691) at a marked distance of the membrane. Mutation of individual amino acids highlighted the importance of Tyr684 and Leu689. The 14-amino acid sequence was grafted onto the p75 neurotrophin receptor and redirected this apical protein to the basolateral cell membrane compartment. Deletion of amino acids 677–695 did not modify the internalization of the FSHR, showing that the basolateral localization signal of the FSHR is not colinear with its internalization signal.

Monoclonal antibodies have been raised against the LH/CG receptor [1] and have allowed to perform immunochemical studies of the receptor in target cells. Three different forms of the LH/CG receptor are physiologically expressed: a mature approximately 85 kDa transmembrane species corresponding to the full length receptor, a approximately 68 kDa high mannose containing species corresponding to a precursor which accumulates inside the cells, and truncated soluble approximately 45-48 kDa molecular weight species corresponding to the variant messanger RNAs generated by alternative splicing. Monoclonal antibodies against the human FSH receptor were also prepared. They allow to observe the existence of two forms of the FSH receptor in the ovaries: a major approximately 87 kDa species corresponding to the mature receptor and a minor approximately 81 kDa species corresponding to a high mannose rich precursor. No variant forms of the receptor corresponding to alternative mRNA transcripts were detected. The transport of hCG was examined in rat testicular microvasculature by electron microscopy and by analyzing the transfer of radiolabeled hormone and antireceptor antibodies. LH/CG receptors were present in endothelial cells and were involved in hormone transcytosis through these cells. Immunocytochemical experiments have shown that the FSH receptor has a polarized expression in the Sertoli cells of the testes whereas the LH/Cg receptor is spread on the surface of thecal granulosa and luteal cells in the ovary and Leydig cells in the testes. To study the mechanism of this polarization FSH, LH and TSH receptors were expressed in polarized MDCK cells. The mechanism of basolateral localization and of transcytosis of the receptors was studied using this model. The effect of hormone, cAMP and agents acting on G proteins was examined.

Gonadotrophin and thyrotrophin receptors belong to a subgroup of G-protein-coupled receptors. These receptors are characterized by a large extracellular domain that is responsible for the binding of the hormone. Soluble receptors, such as some luteinizing hormone receptors, arise from premessenger RNA alternative splicing, or, in the case of thyroid-stimulating hormone (TSH) receptors, by the cleavage and shedding of the ectodomain. Follicle-stimulating hormone and TSH receptors are restricted to the basolateral domain of their target cells. These receptors are also present in endothelial cells of target organ vessels and are involved in hormone transcytosis. Various genetic abnormalities of these receptors have been described.