PHOSPHATIDYLINOSITOL 3-KINASE (PI3K) AS A THERAPEUTIC TARGET IN NSCLC

Deregulated activation of phosphatidylinositol 3-kinase (PI3K) pathway is central to many human malignancies. The functions of this pathway are critical for normal cell metabolism, proliferation, and survival. In lung cancers, the PI3K pathway activity is often aberrantly driven by multiple mutations, including EGFR, KRAS, and PIK3CA. Molecules targeting the PI3K pathway are intensely investigated as potential anti-cancer agents. Although inhibitors of the pathway are currently in clinical trials, rational and targeted use of these compounds, alone or in combination, requires an understanding of isoform-specific activity in context. We sought to identify class IA PI3K enzyme (p110a/PIK3CA, p110b/PIK3CB, p110d/PIK3CD) activities using isoform-specific inhibitors in a lung cancer model system. Treatment of non-small cell lung cancer (NSCLC) cell lines with PIK3CA, PIK3CB, PIK3CD or PIK3CB/D inhibitors resulted in pharmacokinetic and pharmacodynamic responses that frequently tracked with a specific mutation status. Activation of PIK3CA dictated response to the PIK3CA-specific inhibitor while deletion of PTEN phosphatase indicated response to the PIK3CB inhibitor. The PIK3CD isoform-specific inhibitors lacked efficacy in all NSCLC cell lines tested, however treatment at increased concentrations likely provide concurrent inhibition of both PIK3CB/D isoforms improving activity of either agent alone but did not track with a single biomarker. The observed pharmacodynamic and proliferation responses to isoform-specific inhibitors suggested that PI3K isoforms may functionally compensate for loss of another in certain genetic backgrounds. These studies demonstrate unanticipated cellular responses to PI3K isoform inhibition in NSCLC, suggesting that patient populations with specific mutations can benefit from certain isoform-selective inhibitors, or combinations, allowing for rational and targeted clinical use of these agents

A Dose-escalation Study of Recombinant Human Interleukin-18 in Combination With Ofatumumab After Autologous Peripheral Blood Stem Cell Transplantation for Lymphoma

Interleukin-18 (IL-18) is an immunostimulatory cytokine that augments antibody-dependent cellular cytotoxicity mediated by human natural killer cells against antibody-coated lymphoma cells in vitro and that has antitumor activity in animal models. Ofatumumab is a CD20 monoclonal antibody with activity against human B-cell lymphomas. A phase I study of recombinant human (rh) IL-18 given with ofatumumab was undertaken in patients with CD20 lymphoma who had undergone high-dose chemotherapy and autologous peripheral blood stem cell transplantation. Cohorts of 3 patients were given intravenous infusions of ofatumumab 1000 mg weekly for 4 weeks with escalating doses of rhIL-18 as a intravenous infusion weekly for 8 consecutive weeks. Nine male patients with CD20 lymphomas were given ofatumumab in combination with rhIL-18 at doses of 3, 10, and 30 μg/kg. No unexpected or dose-limiting toxicities were observed. The mean reduction from predose levels in the number of peripheral blood natural killer cells after the first rhIL-18 infusion was 91%, 96%, and 97% for the 3, 10, and 30 μg/kg cohorts, respectively. Serum concentrations of interferon-γ and chemokines transiently increased following IL-18 dosing. rhIL-18 can be given in biologically active doses by weekly infusions in combination with ofatumumab after peripheral blood stem cell transplantation to patients with lymphoma. A maximum tolerated dose of rhIL-18 plus ofatumumab was not determined. Further studies of rhIL-18 and CD20 monoclonal antibodies in B-cell malignancies are warranted

Phenylephrine, a common cold remedy active ingredient, suppresses uterine contractions through cAMP signalling

Regulation of uterine contractility is an important aspect of women's health. Phenylephrine, a selective agonist of the α1-adrenoceptor and a potent smooth muscle constrictor, is widely used in women even during pregnancy to relieve cold-related symptoms, to treat postpartum haemorrhoid, and during routine eye exams. We performed isometric tension recordings to investigate the effect of phenylephrine on mouse uterine contractility. Phenylephrine decreased spontaneous and oxytocin-induced contractions in non-pregnant mouse uterine rings and strips with an IC50 of ~1 μM. Prazosin, an inhibitor of α1-adrenoceptor, did not prevent phenylephrine-mediated relaxations. Conversely, ICI118551, an antagonist of β2-adrenoceptors, inhibited phenylephrine relaxation. In the presence of ICI118551, high concentrations (>30 μM) of phenylephrine caused mouse uterine contractions, suggesting that β-adrenoceptor-mediated inhibition interferes with the phenylephrine contractile potential. Phenylephrine-dependent relaxation was reduced in the uterus of pregnant mice. We used primary mouse and human uterine smooth muscle cells (M/HUSMC) to establish the underlying mechanisms. Phenylephrine stimulated large increases in intracellular cAMP in M/HUSMCs. These cAMP transients were decreased when HUSMCs were cultured in the presence of oestrogen and progesterone to mimic the pregnancy milieu. Thus, phenylephrine is a strong relaxant in the non-pregnant mouse uterus, but exhibits diminished effect in the pregnant uterus

Molecular Determinants of the Sensitivity to Gq/11-Phospholipase C-dependent Gating, Gd3+ Potentiation, and Ca2+ Permeability in the Transient Receptor Potential Canonical Type 5 (TRPC5) Channel

Transient receptor potential canonical type 5 (TRPC5) is a Ca2+-permeable cation channel that is highly expressed in the brain and is implicated in motor coordination, innate fear behavior, and seizure genesis. The channel is activated by a signal downstream of the G-protein-coupled receptor (GPCR)-Gq/11-phospholipase C (PLC) pathway. In this study we aimed to identify the molecular mechanisms involved in regulating TRPC5 activity. We report that Arg-593, a residue located in the E4 loop near the TRPC5 extracellular Gd3+ binding site, is critical for conferring the sensitivity to GPCR-Gq/11-PLC-dependent gating on TRPC5. Indeed, guanosine 5'-O-(thiotriphosphate) and GPCR agonists only weakly activate the TRPC5R593A mutant, whereas the addition of Gd3+ rescues the mutant's sensitivity to GPCR-Gq/11-PLC-dependent gating. Computer modeling suggests that Arg-593 may cross-bridge the E3 and E4 loops, forming the "molecular fulcrum." While validating the model using site-directed mutagenesis, we found that the Tyr-542 residue is critical for establishing a functional Gd3+ binding site, the Tyr-541 residue participates in fine-tuning Gd3+-sensitivity, and that the Asn-584 residue determines Ca2+ permeability of the TRPC5 channel. This is the first report providing molecular insights into the molecular mechanisms regulating the sensitivity to GPCR-Gq/11-PLC-dependent gating of a receptor-operated channel

Small-molecule CaVα1⋅CaVβ antagonist suppresses neuronal voltage-gated calcium-channel trafficking

Extracellular calcium flow through neuronal voltage-gated CaV2.2 calcium channels converts action potential-encoded information to the release of pronociceptive neurotransmitters in the dorsal horn of the spinal cord, culminating in excitation of the postsynaptic central nociceptive neurons. The CaV2.2 channel is composed of a pore-forming α1 subunit (CaVα1) that is engaged in protein-protein interactions with auxiliary α2/δ and β subunits. The high-affinity CaV2.2α1⋅CaVβ3 protein-protein interaction is essential for proper trafficking of CaV2.2 channels to the plasma membrane. Here, structure-based computational screening led to small molecules that disrupt the CaV2.2α1⋅CaVβ3 protein-protein interaction. The binding mode of these compounds reveals that three substituents closely mimic the side chains of hot-spot residues located on the α-helix of CaV2.2α1 Site-directed mutagenesis confirmed the critical nature of a salt-bridge interaction between the compounds and CaVβ3 Arg-307. In cells, compounds decreased trafficking of CaV2.2 channels to the plasma membrane and modulated the functions of the channel. In a rodent neuropathic pain model, the compounds suppressed pain responses. Small-molecule α-helical mimetics targeting ion channel protein-protein interactions may represent a strategy for developing nonopioid analgesia and for treatment of other neurological disorders associated with calcium-channel trafficking

PreImplantation Factor (PIF) correlates with early mammalian embryo development-bovine and murine models

<p>Abstract</p> <p>Background</p> <p>PreImplantation Factor (PIF), a novel peptide secreted by viable embryos is essential for pregnancy: PIF modulates local immunity, promotes decidual pro-adhesion molecules and enhances trophoblast invasion. To determine the role of PIF in post-fertilization embryo development, we measured the peptide's concentration in the culture medium and tested endogenous PIF's potential trophic effects and direct interaction with the embryo.</p> <p>Methods</p> <p>Determine PIF levels in culture medium of multiple mouse and single bovine embryos cultured up to the blastocyst stage using PIF-ELISA. Examine the inhibitory effects of anti-PIF-monoclonal antibody (mAb) added to medium on cultured mouse embryos development. Test FITC-PIF uptake by cultured bovine blastocysts using fluorescent microscopy.</p> <p>Results</p> <p>PIF levels in mouse embryo culture medium significantly increased from the morula to the blastocyst stage (ANOVA, P = 0.01). In contrast, atretic embryos medium was similar to the medium only control. Detectable - though low - PIF levels were secreted already by 2-cell stage mouse embryos. In single bovine IVF-derived embryos, PIF levels in medium at day 3 of culture were higher than non-cleaving embryos (control) (P = 0.01) and at day 7 were higher than day 3 (P = 0.03). In non-cleaving embryos culture medium was similar to medium alone (control). Anti-PIF-mAb added to mouse embryo cultures lowered blastocyst formation rate 3-fold in a dose-dependent manner (2-way contingency table, multiple groups, X2; P = 0.01) as compared with non-specific mouse mAb, and medium alone, control. FITC-PIF was taken-up by cultured bovine blastocysts, but not by scrambled FITC-PIF (control).</p> <p>Conclusions</p> <p>PIF is an early embryo viability marker that has a direct supportive role on embryo development in culture. PIF-ELISA use to assess IVF embryo quality prior to transfer is warranted. Overall, our data supports PIF's endogenous self sustaining role in embryo development and the utility of PIF- ELISA to detect viable embryos in a non-invasive manner.</p

Preimplantation factor negates embryo toxicity and promotes embryo development in culture

Preimplantation factor (PIF) is secreted by viable mammalian embryos and promotes implantation and trophoblast invasion. Whether PIF also has a direct protective or promoting effect on the developing embryo in culture is unknown. This study examined the protective effects of synthetic PIF (sPIF) on embryos cultured with embryo toxic serum (ETS) from recurrent pregnancy loss patients (n = 14), by morphological criteria at 72 h of culture, and determined sPIF-promoting effect on singly cultured bovine IVF embryo development. sPIF negated the ETS-induced effect by increasing mouse blastocyst rate versus other embryonic stages (odds ratio (OR) 2.01, 95% confidence intervals (CI) 1.14-3.55, chi-squared = 12.74, P = 0.002), increased blastocyst rate (39.0% versus 23.7% ETS alone) and lowered embryo demise rate (11.0% versus 28.8%, OR 0.24, 95% CI 0.11-0.54), which was not replicated by scrambled PIF or the control. sPIF added to bovine embryos for 3 days promoted development at day 7 of culture (11% versus 0%, chi-squared = 4.0, P = 0.045). In conclusion, sPIF prevented embryo demise caused by exposure to ETS and promoted development of singly cultured bovine IVF embryos following short-term exposure. sPIF-based therapy for reducing recurrent pregnancy loss and improving lagging cultured IVF embryo development should be explored. Preimplantation factor (PIF) is a novel peptide, secreted by the embryo at the 2-cell stage. It is present throughout viable pregnancy but absent in non-viable pregnancy. PIF has essential immune-modulatory, acceptance and maintenance effects on the maternal system for embryo implantation, creating the necessary environment in the uterus and helping better embedding of the placenta. Herein we investigate in two complementary models whether PIF can help embryos survive under adverse conditions, protect against recurrent pregnancy loss and promote lagging embryo development. PIF\u27s synthetic analogue (sPIF) is available at suitable purity for human use and its use is proposed in physiological range doses. sPIF administration strongly suggested a targeted and safe treatment regimen, successfully negating the toxicity present in the blood (serum) of patients suffering from recurrent miscarriages in the relevant pooled mouse embryo model used. sPIF showed an equally important beneficial effect to prevent suboptimally developed embryo demise and improve their development. In a (single bovine) IVF model, highly relevant for low-responder human embryos that produce low-quality but viable embryos, sPIF increased embryo survival under adverse conditions. To document sPIF\u27s specific effect, an inactive, same-composition (scrambled) protein was tested, which showed no effect. To evidence sPIF\u27s safety and unique needs-based action, high concentrations added to optimally cultured embryos equally showed no effect. We conclude that sPIF administration may specifically reduce cases of repeat miscarriage and improve viable but suboptimal embryo survival. These added observations help complete PIF\u27s overall crucial function in accommodating and facilitating successful pregnancy. © 2011, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved

The Role of MDM2 in Promoting Genome Stability versus Instability

In cancer, the mouse double minute 2 (MDM2) is an oncoprotein that contributes to the promotion of cell growth, survival, invasion, and therapeutic resistance. The impact of MDM2 on cell survival versus cell death is complex and dependent on levels of MDM2 isoforms, p53 status, and cellular context. Extensive investigations have demonstrated that MDM2 protein–protein interactions with p53 and other p53 family members (p63 and p73) block their ability to function as transcription factors that regulate cell growth and survival. Upon genotoxic insults, a dynamic and intricately regulated DNA damage response circuitry is activated leading to release of p53 from MDM2 and activation of cell cycle arrest. What ensues following DNA damage, depends on the extent of DNA damage and if the cell has sufficient DNA repair capacity. The well-known auto-regulatory loop between p53-MDM2 provides an additional layer of control as the cell either repairs DNA damage and survives (i.e., MDM2 re-engages with p53), or undergoes cell death (i.e., MDM2 does not re-engage p53). Furthermore, the decision to live or die is also influenced by chromatin-localized MDM2 which directly interacts with the Mre11-Rad50-Nbs1 complex and inhibits DNA damage-sensing giving rise to the potential for increased genome instability and cellular transformation