Phosphoinositide-specific Phospholipase C Research Articles

Proinflammatory Signaling Cascades of Periodontopathic Oral Pathogen <i>Porphyromonas gingivalis</i>

Porphyromonas gingivalis, is the most prominent member of the bacteria flora associated with pathogenesis of periodontitis, a chronic inflammatory disease resulting in tooth loss. The extent of oral mucosal reaction to P. gingivalis invasion relays heavily on Toll-like receptors (TLRs) that recognize structurally common motifs of pathogens and initiate antibacterial responses. Among the virulence factors of P. gingivalis implicated in TLRs activation and triggering inflammatory responses leading to the development of periodontitis is the bacterium cell-wall lipopolysaccharide (LPS). The engagement by the LPS of oral mucosal TLR4 leads to initiation of signaling events characterized by the activation of mitogen-activated protein kinase (MAPK) and IκB-kinase complex (IKK) cascades, induction of phosphoinositide-specific phospholipase C (PLC)/protein kinase C (PKC)/PI3K pathway, up-regulation in TGF-α ectodomain shedding and EGFR transactivation, and the amplification of proinflammatory signals by spleen tyrosine kinase (Syk). These events, in turn, exert their control over transcription factors implicated in the induction of the expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) genes that lead to up-regulation in the inflammatory mediators, PGE2 and NO. The systems involved in transcription factors activation, furthermore, remain under additional regulatory control through S-nitrosylation. Moreover, the LPS-induced TLR4 activation provides a docking site for Syk, the activation of which leads to amplification of the inflammatory signals by affecting transcription factors activation and their assembly to transcriptional complexes. Interestingly, the extent of oral mucosal inflammatory response to P. gingivalis remains under modulatory influence by two biologically active peptide hormones, leptin and ghrelin. Therefore, the presence of these multifunctional peptides in oral mucosa and saliva may be of significance in countering the destructive consequences of P. gingivalis—induced chronic mucosal inflammation that characterizes periodontitis.

The Crucial Roles of Enzymes Belonging to Signal Transduction Pathways. Old Molecules as New Therapy Molecular Targets. Approaches, Perspectives and Criticisms

The crucial roles of Enzymes belonging to signal transduction pathways. Old molecules as new therapy molecular targets. Approaches, perspectives and criticisms. Since last ten years the observation that most of the human diseases can be considered “signaling diseases” was corroborated by many reports. In this perspective, increasing evidences demonstrated a crucial role for “signaling enzymes”, the wide number of enzymes involved in specific steps of signal transduction pathways. Many different families of enzymes are involved in a variety of signal transduction pathways. In a signaling system, enzymes are main actors which play important roles, regulating the metabolism and concentration of further signaling molecules. Thus, different families of enzymes regulate a wide number of cell or tissue activities. As one example, enzymes belonging to the Phosphoinositide-specific Phospholipase C (PLC) family were reported to be involved in many key functions such as intra-cellular signaling, membrane trafficking, nuclear signaling based on lipid hydrolysis in response to a wide panel of stimuli, including growth factors, hormones, and neurotransmitters, that act on specific receptors localized at the plasma membrane.

Phosphoinositide-specific phospholipase C\u03b31 inhibition induces autophagy in human colon cancer and hepatocellular carcinoma cells

Phosphoinositide-specific phospholipase C (PLC) γ1 has been reported to be involved in cancer cell proliferation and metastasis. However, whether PLCγ1 modulates autophagy and the underlying mechanism remains unclear. Here, we investigated the relationship between PLCγ1 and autophagy in the human colon cancer cell line HCT116 and hepatocellular carcinoma cell line HepG2. The results indicated that PLCγ1 inhibition via lentivirus-mediated transduction with shRNA/PLCγ1 or transient transfection with pRK5-PLCγ1 (Y783A) vector increased LC3B-II levels and the number of autophagic vacuoles and decreased p62 levels. Addition of an autophagy inhibitor led to LC3B and p62 accumulation. Furthermore, AMPK activation promoted the autophagy induced by PLCγ1 inhibition by blocking the FAK/PLCγ1 axis. In addition, PLCγ1 inhibition either blocked the mTOR/ULK1 axis or enhanced dissociation of the Beclin1-IP3R-Bcl-2 complex to induce autophagy. Taken together, our findings revealed that PLCγ1 inhibition induced autophagy and the FAK/PLCγ1 axis is a potential downstream effector of the AMPK activation-dependent autophagy signalling cascade. Both blockade of the mTOR/ULK1 axis and dissociation of the Beclin1-IP3R-Bcl-2 complex contributed to the induction of autophagy by PLCγ1 inhibition. Consequently, these findings provide novel insight into autophagy regulation by PLCγ1 in colon cancer and hepatocellular carcinoma cells.

Phospholipase C2 Affects MAMP-Triggered Immunity by Modulating ROS Production.

The activation of phosphoinositide-specific phospholipase C (PI-PLC) is one of the earliest responses triggered by the recognition of several microbe-associated molecular patterns (MAMPs) in plants. The Arabidopsis (Arabidopsis thaliana) PI-PLC gene family is composed of nine members. Previous studies suggested a role for PLC2 in MAMP-triggered immunity, as it is rapidly phosphorylated in vivo upon treatment with the bacterial MAMP flg22. Here, we analyzed the role of PLC2 in plant immunity using an artificial microRNA to silence PLC2 expression in Arabidopsis. We found that PLC2-silenced plants are more susceptible to the type III secretion system-deficient bacterial strain Pseudomonas syringae pv tomato (Pst) DC3000 hrcC- and to the nonadapted pea (Pisum sativum) powdery mildew Erysiphe pisi However, PLC2-silenced plants display normal susceptibility to virulent (Pst DC3000) and avirulent (Pst DC3000 AvrRPM1) P. syringae strains, conserving typical hypersensitive response features. In response to flg22, PLC2-silenced plants maintain wild-type mitogen-activated protein kinase activation and PHI1, WRKY33, and FRK1 immune marker gene expression but have reduced reactive oxygen species (ROS)-dependent responses such as callose deposition and stomatal closure. Accordingly, the generation of ROS upon flg22 treatment is compromised in the PLC2-defficient plants, suggesting an effect of PLC2 in a branch of MAMP-triggered immunity and nonhost resistance that involves early ROS-regulated processes. Consistently, PLC2 associates with the NADPH oxidase RBOHD, suggesting its potential regulation by PLC2.

Gibberellic acid promoting phytic acid degradation in germinating soybean under calcium lactate treatment.

Phytic acid as a phosphorus storage vault provides phosphorus for plant development. It is an anti-nutritional factor for humans and some animals. However, its degradation products lower inositol phosphates have positive effects on human health. In this study, the effect of gibberellic acid (GA) on phytic acid degradation under calcium lactate (Ca) existence was investigated. The results showed that Ca + GA treatment promoted the growth status, hormone metabolism and phytic acid degradation in germinating soybean. At the same time, the availability of phosphorus, the activity of phytic acid degradation-associated enzyme and phosphoinositide-specific phospholipase C (PI-PLC) increased. However, the relative genes expression of phytic acid degradation-associated enzymes did not vary in accordance with their enzymes activity. The results revealed that GA could mediate the transport and function of calcium and a series of physiological and biochemical changes to regulate phytic acid degradation of soybean sprouts. © 2017 Society of Chemical Industry.

Role of LPS-elicited signaling in triggering gastric mucosal inflammatory responses to H. pylori: modulatory effect of ghrelin.

Infection with Helicobacter pylori is a primary culprit in the etiology of gastric disease, and its cell-wall lipopolysaccharide (LPS) is recognized as a potent endotoxin responsible for triggering a pattern of the mucosal inflammatory responses. The engagement by the LPS of gastric mucosal Toll-like receptor 4 (TLR4) leads to initiation of signal transduction events characterized by the activation of mitogen-activated protein kinase (MAPK) cascade, induction of phosphoinositide-specific phospholipase C (PLC)/protein kinase C (PKC)/phosphatidylinositol 3-kinase (PI3K) pathway, and up-regulation in Src/Akt. These signaling events in turn exert their influence over H. pylori-elicited excessive generation of NO and PGE2 caused by the disturbances in nitric oxide synthase and cyclooxygenase isozyme systems, increase in epidermal growth factor receptor transactivation, and the induction in matrix metalloproteinase-9 (MMP-9) release. Interestingly, the extent of gastric mucosal inflammatory response to H. pylori is influenced by a peptide hormone, ghrelin, the action of which relays on the growth hormone secretagogue receptor type 1a (GHS-R1a)-mediated mobilization of G-protein dependent transduction pathways. Yet, the signals triggered by TLR-4 activation as well as those arising through GHS-R1a stimulation converge at MAPK and PLC/PKC/PI3K pathways that form a key integration node for proinflammatory signals generated by H. pylori LPS as well as for those involved in modulation of inflammation by ghrelin. Hence, therapeutic targeting these signals' convergence and integration node could provide a novel and attractive opportunities for developing more effective treatments of H. pylori-related gastric disease.

Activation of dopamine D1 receptors enhances the temporal summation and excitability of rat retinal ganglion cells

Dopamine (DA), an important neurotransmitter and neuromodulator, plays important roles in neuronal physiological functions by activating G-protein-coupled DA D1 and/or D2 receptors. Previous studies have demonstrated that D1 receptors are functionally expressed in retinal neurons and glial cells, including ganglion cells. In this study, we explored the effects of D1 receptor activation on retinal ganglion cell (RGC) temporal summation and excitability in rat retinal slices using electrophysiological techniques. Bath application of the selective D1 receptor agonist SKF81297 increased the ratio of excitatory postsynaptic potentials (EPSPs) (EPSP5/EPSP1) within an EPSP train evoked by a train stimulation (five current pulses at 40Hz), which was blocked by co-application of SCH23390, a specific D1 receptor antagonist. Ba2+, an inwardly rectifying K+ channel (Kir) blocker, significantly suppressed the SKF81297-induced effect, whereas ZD7288, a specific hyperpolarization-activated cation current (Ih) blocker, showed a moderate inhibitory effect. The cAMP/protein kinase A (PKA) signaling pathway, but not phosphoinositide-specific phospholipase C (PI-PLC), mediated the SKF81297-induced modulation of EPSP temporal summation. Further experiments showed that SKF81297 suppressed Ba2+-sensitive Kir currents in RGCs. Additionally, SKF81297 increased the spontaneous firing frequency of RGCs, and caused depolarization of the cells with or without the presence of synaptic receptor blockers. In contrast, SKF81297 did not significantly change the frequency of miniature excitatory postsynaptic currents (mEPSCs) recorded in RGCs. Our results indicate that D1 receptor activation enhances the temporal summation of RGCs mainly by suppressing Kir currents through the cAMP/PKA signaling pathway, thus increasing the excitability of rat RGCs.

Arabidopsis phosphoinositide-specific phospholipase C 4 negatively regulates seedling salt tolerance.

Previous physiological and pharmacological studies have suggested that the activity of phosphoinositide-specific phospholipase C (PI-PLC) plays an important role in regulating plant salt stress responses by altering the intracellular Ca2+ concentration. However, the individual members of plant PLCs involved in this process need to be identified. Here, the function of AtPLC4 in the salt stress response of Arabidopsis seedlings was analysed. plc4 mutant seedlings showed hyposensitivity to salt stress compared with Col-0 wild-type seedlings, and the salt hyposensitive phenotype could be complemented by the expression of native promoter-controlled AtPLC4. Transgenic seedlings with AtPLC4 overexpression (AtPLC4 OE) exhibited a salt-hypersensitive phenotype, while transgenic seedlings with its inactive mutant expression (AtPLC4m OE) did not exhibit this phenotype. Using aequorin as a Ca2+ indicator in plc4 mutant and AtPLC4 OE seedlings, AtPLC4 was shown to positively regulate the salt-induced Ca2+ increase. The salt-hypersensitive phenotype of AtPLC4 OE seedlings was partially rescued by EGTA. An analysis of salt-responsive genes revealed that the transcription of RD29B, MYB15 and ZAT10 was inversely regulated in plc4 mutant and AtPLC4 OE seedlings. Our findings suggest that AtPLC4 negatively regulates the salt tolerance of Arabidopsis seedlings, and Ca2+ may be involved in regulating this process.

Heme Assimilation in Schizosaccharomyces pombe Requires Cell-surface-anchored Protein Shu1 and Vacuolar Transporter Abc3

The Schizosaccharomyces pombe shu1+ gene encodes a cell-surface protein required for assimilation of exogenous heme. In this study, shaving experiments showed that Shu1 is released from membrane preparations when spheroplast lysates are incubated with phosphoinositide-specific phospholipase C (PI-PLC). Shu1 cleavability by PI-PLC and its predicted hydropathy profile strongly suggested that Shu1 is a glycosylphosphatidylinositol-anchored protein. When heme biosynthesis is selectively blocked in hem1Δ mutant cells, the heme analog zinc mesoporphyrin IX (ZnMP) first accumulates into vacuoles and then subsequently, within the cytoplasm in a rapid and Shu1-dependent manner. An HA4-tagged shu1+ allele that retained wild-type function localizes to the cell surface in response to low hemin concentrations, but under high hemin concentrations, Shu1-HA4 re-localizes to the vacuolar membrane. Inactivation of abc3+, encoding a vacuolar membrane transporter, results in hem1Δ abc3Δ mutant cells being unable to grow in the presence of hemin as the sole iron source. In hem1Δ abc3Δ cells, ZnMP accumulates primarily in vacuoles and does not sequentially accumulate in the cytosol. Consistent with a role for Abc3 as vacuolar hemin exporter, results with hemin-agarose pulldown assays showed that Abc3 binds to hemin. In contrast, an Abc3 mutant in which an inverted Cys-Pro motif had been replaced with Ala residues fails to bind hemin with high affinity. Taken together, these results show that Shu1 undergoes rapid hemin-induced internalization from the cell surface to the vacuolar membrane and that the transporter Abc3 participates in the mobilization of stored heme from the vacuole to the cytosol.

The structure of a calcium-dependent phosphoinositide-specific phospholipase C from Pseudomonas sp. 62186, the first from a Gram-negative bacterium.

Bacterial phosphoinositide-specific phospholipases C (PI-PLCs) are the smallest members of the PI-PLC family, which includes much larger mammalian enzymes responsible for signal transduction as well as enzymes from protozoan parasites, yeast and plants. Eukaryotic PI-PLCs have calcium in the active site, but this is absent in the known structures of Gram-positive bacteria, where its role is instead played by arginine. In addition to their use in a number of industrial applications, the bacterial enzymes attract special interest because they can serve as convenient models of the catalytic domains of eukaryotic enzymes for in vitro activity studies. Here, the structure of a PI-PLC from Pseudomonas sp. 62186 is reported, the first from a Gram-negative bacterium and the first of a native bacterial PI-PLC with calcium present in the active site. Solution of the structure posed particular problems owing to the low sequence identity of available homologous structures. Its dependence on calcium for catalysis makes this enzyme a better model for studies of the mammalian PI-PLCs than the previously used calcium-independent bacterial PI-PLCs.

Arabidopsis phosphatidylinositol-phospholipase C2 (PLC2) is required for female gametogenesis and embryo development.

AtPLC2 is an essential gene in Arabidopsis, since it is required for female gametogenesis and embryo development. AtPLC2 might play a role in cell division during embryo-sac development and early embryogenesis. Phosphoinositide-specific phospholipase C (PI-PLC) plays an important role in signal transduction during plant development and in the response to various biotic- and abiotic stresses. The Arabidopsis PI-PLC gene family is composed of nine members, named PLC1 to PLC9. Here, we report that PLC2 is involved in female gametophyte development and early embryogenesis. Using two Arabidopsis allelic T-DNA insertion lines with different phenotypic penetrations, we observed both female gametophytic defects and aberrant embryos. For the plc2-1 mutant (Ws background), no homozygous plants could be recovered in the offspring from self-pollinated plants. Nonetheless, plc2-1 hemizygous mutants are affected in female gametogenesis, showing embryo sacs arrested at early developmental stages. Allelic hemizygous plc2-2 mutant plants (Col-0 background) present reduced seed set and embryos arrested at the pre-globular stage with abnormal patterns of cell division. A low proportion (0.8%) of plc2-2 homozygous mutants was found to escape lethality and showed morphological defects and disrupted megagametogenesis. PLC2-promoter activity was observed during early megagametogenesis, and after fertilization in the embryo proper. Immunolocalization studies in early stage embryos revealed that PLC2 is restricted to the plasma membrane. Altogether, these results establish a role for PLC2 in both reproductive- and embryo development, presumably by controlling mitosis and/or the formation of cell-division planes.

Role of Nuclear Inositide Signalling and microRNA Signature in Myelodysplastic Syndromes during Azacitidine and Lenalidomide Therapy

Phosphoinositide-specific phospholipase C (PI-PLC) gamma1 is involved in erythroid differentiation, via activation of the Akt/PI-PLCgamma1 pathway, and its increase has been associated with erythropoiesis in MDS cells. On the other hand, PI-PLCbeta1 is a nuclear inositide-dependent enzyme implicated in the regulation of hematopoietic differentiation. Interestingly, PI-PLCbeta1 increase plays an important predictive role in the response of MDS cells to epigenetic therapy. Indeed, PI-PLCbeta1 promoter is specifically hypomethylated by azacitidine, a demethylating agent that is clinically used in MDS to improve patients' overall survival and delay the AML evolution. Moreover, azacitidine is currently studied in combination with lenalidomide, to sustain both myeloid and erythroid lineages and balance MDS cell proliferation and differentiation. However, the molecular effects of this combination therapy on inositide signalling pathways and microRNA expression are still unclear.This study included 44 patients diagnosed with high-risk MDS who were given azacitidine and lenalidomide. Patients were considered clinically evaluable after at least 6 cycles of treatment. Molecular analyses were performed at baseline and during the therapy. At first, Real-Time PCR and immunocytochemical experiments were performed to determine PI-PLCbeta1 and PI-PLCgamma1 expression. Then, we also carried out cell cycle analyses and studied both PI-PLCbeta1 methylation status and the expression of erythroid-specific molecules, i.e. Globin genes. On the other hand, to further investigate the effect of the combination therapy on epigenetic mechanisms, we analyzed microRNA expression at baseline and during the treatment. In particular, we started by comparing the 4th cycle of the therapy to baseline, and in case of significant differences, for responder patients, we carried out microRNA profiling at the 8th cycle of the therapy or during the follow-up.Our study included 44 patients, but only 28 subjects were clinically evaluable, with an overall response rate of 78.6% (22/28 cases). At a molecular level, a significant increase of PI-PLCbeta1 expression was associated with a favourable clinical response to the combination therapy. Moreover, responder cases also showed an increased expression of Beta-Globin and PI-PLCgamma1, hinting at a specific contribution of lenalidomide on erythroid activation. On the other hand, the frequent demethylation of PI-PLCbeta1 promoter in responder cases could be specifically linked to azacitidine. Furthermore, MDS cells treated with azacitidine and lenalidomide not only showed an increased G0/G1 phase of cell cycle, but also microRNA expression was affected. In fact, responder and non responder cases showed a specific molecular pattern of microRNAs and, interestingly, some of these microRNAs can target or are strictly associated with inositide signalling pathways.Our results show that the combination of azacitidine and lenalidomide in high-risk MDS patients can be important to induce PI-PLCbeta1, and possibly PI-PLCgamma1. These enzymes can regulate cell cycle, myeloid and erythroid differentiation, thus improving peripheral cytopenia. On the other hand, a specific microRNA signature is important to make a molecular distinction between responder and non responder cases, so that their expression or interactions, possibly with PI-PLCs or other nuclear inositides, can be important to disclose new mechanisms in MDS pathogenesis and identify new predictive markers for the assessment of the response to azacitidine and lenalidomide therapy. DisclosuresGobbi:Novartis: Consultancy, Research Funding; Mundipharma: Consultancy, Research Funding; Roche: Honoraria; Janssen: Consultancy, Honoraria; Gilead: Honoraria; Celgene: Consultancy; Takeda: Consultancy. Finelli:Novartis: Other: Speaker fees; Celgene: Other: Speaker fees; Celgene: Research Funding.

IP3 Mediates Nitric Oxide–Guanosine 3′,5′-Cyclic Monophosphate (NO-cGMP)-Induced Isoflavone Accumulation in Soybean Sprouts under UV-B Radiation

In this study, to investigate the role of inositol 1,4,5-trisphosphate (IP3) in nitric oxide-guanosine 3',5'-cyclic monophosphate (NO-cGMP)-induced isoflavone accumulation in soybean sprouts under UV-B radiation, the sprouts were treated with donors and inhibitors of NO and cGMP as well as IP3 inhibitor. Results showed that NO, with cGMP as a second messenger, stimulates IP3 accumulation under UV-B radiation. Consistent with the increase in IP3 content, the up-regulation of gene and protein expression of phosphoinositide-specific phospholipase C (PI-PLC) in response to sodium nitroprusside (SNP) (exogenous NO donor) and 8-Br-cGMP (cGMP analogue) was also observed. In addition, protein kinase G (PKG) participated in NO-cGMP-induced IP3 production. IP3 induced by the NO-cGMP pathway was involved in isoflavone synthesis by elevating the activity and gene and protein expressions of chalcone synthase (CHS) and isoflavone synthase (IFS). Overall, IP3 mediates NO-cGMP-induced isoflavone accumulation in soybean sprouts under UV-B stress.

Identification of anticancer agents based on the thieno[2,3-b]pyridine and 1H-pyrazole molecular scaffolds

Structural similarity search of commercially available analogues of thieno[2,3-b]pyridine and 1H-pyrazole derivatives, known anticancer agents, resulted in 717 hits. These were docked into the phosphoinositide specific-phospholipase C (PLC) binding pocket, the putative target of the compounds, to further focus the selection. Thirteen derivatives of the thieno[2,3-b]pyridines were identified and tested against the NCI60 panel of human tumour cell lines. The most active derivative 1 was most potent against the MDA-MB-435 melanoma cell line with GI50 at 30nM. Also, it was found that a piperidine moiety is tolerated on the thieno[2,3-b]pyridine scaffold with GI50=296nM (MDA-MB-435) for derivative 10 considerably expanding the structure activity relationship for the series. For the 1H-pyrazoles four derivatives were identified using the in silico approach and additionally ten were synthesised with various substituents on the phenyl moiety to extend the structural activity relationship but only modest anticancer activity was found.

Identification and characterization of genes associated with thermo-tolerance using virus induced gene silencing in Nicotiana benthamiana

Crop acclimatization to elevated temperature conditions often demands transcriptional reprogramming of an array of genes to evoke protection from heat stress. Previously, we addressed such question in potato and identified GLP1 (Germin-like protein 1), nsLTP (Non specific lipid transfer protein), PI-PLC (phosphoinositide-specific phospholipase-c), CHP (Conserved hypothetical protein) and RPL4 (60 S Ribosomal L4/L1 protein) as candidate thermo-tolerant genes. Herein, Nicotiana benthamiana knockdown lines were developed to study their functional relevance in high-temperature stress using virus induced gene silencing (VIGS). Among all the VIGS lines tested, silencing of GLP1 and CHP showed significant reduction (more than 75 %) in their endogenous transcript levels, whereas the expression of nsLTP, PI-PLC and RPL4 was reduced by 50 %. Thus suggesting the silencing of endogenous target gene was successful using VIGS. Among those genes tested, VIGS knockdown lines generated against GLP1 and nsLTP produced a very strong hypersensitive phenotype under gradual heat stress and sudden heat shock conditions. Knockdown of GLP1 and nsLTP also showed higher chlorophyll degradation and less cell viability upon stress compared with mock plants. Interestingly, down-regulation of PI-PLC plants showed thermo-sensitive to sudden heat shock, whereas CHP and RPL4 silenced plants showed relatively less sensitive phenotypes. Most importantly, silencing of GLP1 and nsLTP genes compromised acclimation induced H2O2 accumulation, which subsequently failed to activate reactive oxygen species (ROS) scavenging signaling pathways that controlling transcription of antioxidant and other heat stress related genes. These data suggest that GLP1 or nsLTP dependent H2O2 production is required for acquiring thermo-tolerance mediated via activation of ROS-related genes. These analyses will aid in an effort to engineer broad spectrum thermo-tolerance in economically important crop.

Association of Azacitidine and Lenalidomide (Combined vs Sequential Treatment) in High-Risk Myelodysplastic Syndromes. Final Results of a Randomized Phase II Multicenter Study

Introduction. Azacitidine (AZA) is able to induce hematologic responses in 50-60 % of patients (pts) with Myelodysplastic Syndromes (MDS) and moreover to prolong survival in higher risk MDS pts. Recently, several studies have evaluated the efficacy and safety of combining, in high-risk MDS pts, AZA with Lenalidomide (LEN), either administered concurrently (Sekeres, 2010; 2012), or sequentially (Platzbecker, 2013), in both cases showing promising results, although in a limited number of pts. The aim of this study was to evaluate the efficacy and safety of the combination vs the sequential use of AZA and LEN in high-risk MDS pts (IPSS score risk: High or INT-2). Primary endpoint: ORR, defined as the Rate of Complete Remission (CR), Partial Remission (PR), Marrow Complete Remission (mCR), and Hematological Improvement (HI), following the International Working Group (IWG) criteria (Cheson, 2006).Methods. This is a randomized, phase II, multicenter, open label study, including pts with MDS (WHO 2008 classification) with International Prognostic Scoring System (IPSS) risk High or Intermediate-2, without previous treatment with AZA or LEN. ARM 1 (combined treatment): AZA: 75 mg/m2/day (days 1-5) I.C. + LEN: 10 mg/day (days 1-21), orally, every 4 weeks. ARM 2 (sequential treatment): AZA: 75 mg/m2/day (days 1-5) I.C. + LEN: 10 mg/day (days 6-21), orally, every 4 weeks. The induction treatment was planned for 8 cycles (32 weeks). For responder patients (CR, PR, mCR, or HI) the same treatment was continued until disease progression or unacceptable toxicity. A sample size of 44 pts was planned.Results. From March 2013, 44 pts (27 males), with a median age of 72 (48-83 yrs) were enrolled, from 13 hematologic italian Centers. At baseline, WHO diagnosis was: RCMD: 2 pts; RCMD-RS: 1 pt ; RAEB-1: 11 pts; RAEB-2: 30 pts; IPSS risk was: Intermediate-2: 31 pts; High: 9 pts; not determined (N.D.) (because of lack of cytogenetic data): 2 pts. (all with RAEB-2). In 2 pts IPSS risk was Intermediate-1, but they were enrolled because of severe thrombocytopenia and neutropenia, respectively. IPSS-R risk was: intermediate: 8 pts; High: 16 pts; Very-High: 18 pts; N.D.: 2 pts; IPSS cytogenetic risk was: Good: 17 pts; Intermediate: 11 pts; Poor: 14 pts; N.D.: 2 pts. 21 pts were randomly assigned to ARM 1, and 23 pts to ARM 2. At the time of this analysis, enrolment of the planned 44 pts was completed. 34/44 pts (77.3%) completed ≥ 6 cycles of treatment, and are evaluable for response. The remaining 10 pts (4 in ARM 1 and 6 in ARM 2) are not evaluable for response, as they discontinued treatment before completing the 6th cycle because of adverse events (6 pts, 13.6%), consent withdrawal (2 pts, 4.5%) or medical decision (2 pts, 4.5%), respectively. Treatment was given for a median of 8 (1-28) cycles; in ARM 1: 9 (1-22) cycles, in ARM 2: 8 (1-28) cycles, respectively. Among the 34 pts evaluable for response, 26/34 pts (ORR: 76.5 %) showed a favourable response to treatment. The Best Response achieved was: CR: 8 pts (23.5%), PR: 1 pt (2.9%), mCR: 3 pts (8.8%), HI: 8 pts (23.5%), mCR+HI: 6 pts (17.6%). The remaining 8 pts showed either Stable Disease (SD) (6 pts, 17.6%) or Disease Progression (DP) (2 pts, 5.9%). First Response was detected after a median of 2 (1-8) cycles. Responder pts were: 13/17 (ORR: 76.5 %) in ARM 1 (3 CR; 1 PR; 1 mCR; 4 HI, 4 mCR+HI), and 13/17 (ORR: 76.5 %) in ARM 2 (5 CR; 2 mCR; 4 HI; 2 mCR+HI), respectively. Overall, the median duration of response was 8.5 (2-23) months: 6 (2-19) months in ARM 1; 16 (2-23) months in ARM 2. A grade > 2 non hematologic toxicity was observed in 24/44 (54.5 %) pts (ARM 1: 66.7%; ARM 2: 43.5%). 27/44 pts (61.4 %) (ARM 1: 61.9%; ARM 2: 60.9%) had a dose reduction of LEN because of hematologic or non-hematologic toxicity. 22 pts (50%) died (ARM 1: 47.6%; ARM 2: 52.2%). 14 pts (31.8%) (ARM 1: 23.8%; ARM 2: 39.1%) showed progression to AML. Overall, median survival was 13 (1-28) months; ARM 1: 13 (1-25) months; ARM 2: 14 (2-28) months.Conclusions. Our results confirm the efficacy of both AZA + LEN treatment regimens in high-risk MDS pts. Moreover, at a molecular level, a significant increase of phosphoinositide-specific phospholipase C (PI-PLC) beta1 and/or PI-PLCgamma1 expression was associated with a favourable clinical response to treatment. Responder cases also showed an increase of Beta-globin expression, hinting at a specific contribution of LEN on erythroid activation DisclosuresFinelli:Janssen: Other: Speaker; Novartis: Other: Speaker; Celgene: Other: Speaker, Research Funding. Visani:Celgene: Research Funding. Cavo:Janssen-Cilag, Celgene, Amgen, BMS: Honoraria.

Phosphoinositide-specific phospholipase C in health and disease

Phospholipases are widely occurring and can be found in several different organisms, including bacteria, yeast, plants, animals, and viruses. Phospholipase C (PLC) is a class of phospholipases that cleaves phospholipids on the diacylglycerol (DAG) side of the phosphodiester bond producing DAGs and phosphomonoesters. Among PLCs, phosphoinositide-specific PLC (PI-PLC) constitutes an important step in the inositide signaling pathways. The structures of PI-PLC isozymes show conserved domains as well as regulatory specific domains. This is important, as most PI-PLCs share a common mechanism, but each of them has a peculiar role and can have a specific cell distribution that is linked to a specific function. More importantly, the regulation of PLC isozymes is fundamental in health and disease, as there are several PLC-dependent molecular mechanisms that are associated with the activation or inhibition of important physiopathological processes. Moreover, PI-PLC alternative splicing variants can play important roles in complex signaling networks, not only in cancer but also in other diseases. That is why PI-PLC isozymes are now considered as important molecules that are essential for better understanding the molecular mechanisms underlying both physiology and pathogenesis, and are also potential molecular targets useful for the development of innovative therapeutic strategies.

The dipeptidyl peptidase IV inhibitors vildagliptin and K-579 inhibit a phospholipase C: a case of promiscuous scaffolds in proteins.

The long term side effects of any newly introduced drug is a subject of intense research, and often raging controversies. One such example is the dipeptidyl peptidase-IV (DPP4) inhibitor used for treating type 2 diabetes, which is inconclusively implicated in increased susceptibility to acute pancreatitis. Previously, based on a computational analysis of the spatial and electrostatic properties of active site residues, we have demonstrated that phosphoinositide-specific phospholipase C (PI-PLC) from Bacillus cereus is a prolyl peptidase using in vivo experiments. In the current work, we first report the inhibition of the native activity of PI-PLC by two DPP4 inhibitors - vildagliptin (LAF-237) and K-579. While vildagliptin inhibited PI-PLC at micromolar concentrations, K-579 was a potent inhibitor even at nanomolar concentrations. Subsequently, we queried a comprehensive, non-redundant set of 5000 human proteins (50% similarity cutoff) with known structures using serine protease (SPASE) motifs derived from trypsin and DPP4. A pancreatic lipase and a gastric lipase are among the proteins that are identified as proteins having promiscuous SPASE scaffolds that could interact with DPP4 inhibitors. The presence of such scaffolds in human lipases is expected since they share the same catalytic mechanism with PI-PLC. However our methodology also detects other proteins, often with a completely different enzymatic mechanism, that have significantly congruent domains with the SPASE motifs. The reported elevated levels of serum lipase, although contested, could be rationalized by inhibition of lipases reported here.In an effort to further our understanding of the spatial and electrostatic basis of DPP4 inhibitors, we have also done a comprehensive analysis of all 76 known DPP4 structures liganded to inhibitors till date. Also, the methodology presented here can be easily adopted for other drugs, and provide the first line of filtering in the identification of pathways that might be inadvertently affected due to promiscuous scaffolds in proteins.

Ezrin-related Phosphoinositide pathway modifies RhoA and Rac1 in human osteosarcoma cell lines.

Selected Phosphoinositide-specific Phospholipase C (PI-PLC) enzymes occupy the convergence point of the broad range of pathways that promote Rho and Ras GTPase mediated signalling, which also regulate the activation of ezrin, a member of the ezrin-radixin-moesin (ERM) proteins family involved in the metastatic osteosarcoma spread. Previous studies described that in distinct human osteosarcoma cell lines ezrin networks the PI-PLC with complex interplay controlling the expression of the PLC genes, which codify for PI-PLC enzymes. In the present study, we analyzed the expression and the sub-cellular distribution of RhoA and Rac1 respectively after ezrin silencing and after PI-PLC ε silencing, in order to investigate whether ezrin-RhoGTPAses signalling might involve one or more specific PI-PLC isoforms in cultured 143B and Hs888 human osteosarcoma cell lines. In the present experiments, both ezrin and PLCE gene silencing had different effects upon RhoA and Rac1 expression and sub-cellular localization. Displacements of Ezrin and of RhoA localization were observed, probably playing functional roles.

Dipeptidyl peptidase-IV inhibitors used in type-2 diabetes inhibit a phospholipase C: a case of promiscuous scaffolds in proteins

The long term side effects of any newly introduced drug is a subject of intense research, and often raging controversies. One such example is the dipeptidyl peptidase-IV (DPP4) inhibitor used for treating type 2 diabetes, which is inconclusively implicated in increased susceptibility to acute pancreatitis. Previously, based on a computational analysis of the spatial and electrostatic properties of active site residues, we have demonstrated that phosphoinositide-specific phospholipase C (PI-PLC) from Bacillus cereus is a prolyl peptidase using in vivo experiments. In the current work, we first report the inhibition of the native activity of PI-PLC by two DPP4 inhibitors - vildagliptin (LAF-237) and K-579. While vildagliptin inhibited PI-PLC at micromolar concentrations, K-579 was a potent inhibitor even at nanomolar concentrations. Subsequently, we queried a comprehensive, non-redundant set of 5000 human proteins (50% similarity cutoff) with known structures using serine protease (SPASE) motifs derived from trypsin and DPP4. A pancreatic lipase and a gastric lipase are among the proteins that are identified as proteins having promiscuous SPASE scaffolds that could interact with DPP4 inhibitors. The presence of such scaffolds in human lipases is expected since they share the same catalytic mechanism with PI-PLC. However our methodology also detects other proteins, often with a completely different enzymatic mechanism, that have significantly congruent domains with the SPASE motifs. The reported elevated levels of serum lipase, although contested, could be rationalized by inhibition of lipases reported here. In an effort to further our understanding of the spatial and electrostatic basis of DPP4 inhibitors, we have also done a comprehensive analysis of all 76 known DPP4 structures liganded to inhibitors till date. Also, the methodology presented here can be easily adopted for other drugs, and provide the first line of filtering in the identification of pathways that might be inadvertently affected due to promiscuous scaffolds in proteins.