Additional Mechanisms Of Action Research Articles

Abstract 2611: Effects of chalcones, nicotinamide, and resveratrol on PPAR gamma activation in oral cancer cells

Abstract Several classes of natural products have cancer prevention potential and PPAR gamma is an intriguing chemoprevention target as a nuclear receptor that controls multiple growth and differentiation processes. We have previously tested many classes of natural products in A/J mice and have found that the following chemoprevention compounds had no significant dietary toxicities: inositol, eucalyptol, chalcones, nicotinamides, and resveratrol. We tested these compounds for their capacity to activate PPAR gamma reporter genes in CA-9-22 oral cancer cells. In several repeated experiments we found that Inositol hexaphosphate did not activate PPAR gamma reporter genes at high concentrations of 1-4 mM. We found that eucalyptol, trans-Chalcone, 4 methoxy chalcone, Nicotinic Acid, and Nicotinamide had the capacity for statistically significant activation of PPAR gamma reporter genes in oral cancer cells from 20-60% at concentrations of 1-100 uM. We found that resveratrol was the most potent agent and could activate PPAR gamma reporter genes 2 fold. As a reference, this was about half the activity of the prototypic PPAR gamma activator pioglitazone. We performed additional experiments with resveratrol and found that it could activate the PPAR gamma dependent squamous differentiation gene Involucrin in oral cancer cells and BEAS 2B cells. Additionally, resveratrol significantly decreased BEAS 2 B proliferation from 48-96 hours at concentrations of 10 uM. From this data we conclude that resveratrol has the capacity to activate PPAR gamma in vitro and resveratrol and its derivatives may also function as PPAR gamma activators as an additional mechanism of action. We also conclude that inositol, eucalyptol, chalcones, and nicotinamides do not activate PPAR gamma. Citation Format: Jennifer Diaz, Beverly Wuertz, Art Galbraith, Frank G. Ondrey. Effects of chalcones, nicotinamide, and resveratrol on PPAR gamma activation in oral cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2611.

Abstract 595: Next-generation engineered toxin bodies: CD38, PD-L1 and HER2 targeted ETBs

Abstract Molecular Templates is developing engineered toxin bodies (ETBs), potent recombinant immunotoxins that combine the specificity of an antibody fragment with the powerful direct cytotoxicity of the Shiga-like toxin A subunit to specifically kill target expressing cells. Once delivered to appropriate cells, the Shiga toxin A subunit inhibits protein synthesis and promotes apoptosis of tumor cells. Bacterial or plant derived toxin moieties have the potential to induce an immune response, commonly limiting repeat dosing of immunotoxins. Our next-generation ETB scaffold has been modified to overcome this limitation through genetic engineering to systematically and comprehensively reduce B and CD4+T cell epitopes. Molecular Templates has developed a proprietary epitope class switching technology designed to both reduce the anti-drug response and promote the anti-tumor response by replacing naturally occurring CD4+ T cell epitopes with CD8+ T cell epitopes. We have found that a combination of surface remodeling and epitope class switching combined in one protein results in powerful reductions in the anti-drug response against ETBs after repeat administration. This de-immunized next-generation scaffold retains the potency and specificity of the unmodified ETB. Additionally, the engineering of CD8+ T cell epitopes on the ETB scaffold can allow for foreign antigen presentation in complex with MHC class I on the tumor cell surface. This antigen presentation may recruit activated cytotoxic T lymphocytes (CTLs) to the tumor microenvironment thereby adding an additional mechanism of action to the direct cell kill activity of the ETBs. The second generation ETB scaffold has been combined with multiple target binding domains, including scFvs that target CD38, PD-L1 and HER2. CD38 is a validated target for multiple myeloma, and our CD38 targeted ETB has shown potent activity in vitro and in vivo. Pre-clinical studies have shown effective combination of the CD38 targeted ETB with standard of care agents such as IMiDs. Additionally, we have developed a PD-L1 targeted ETB that can be used against various PD-L1 expressing malignancies including Hodgkin's lymphoma and melanoma. HER2 is a well characterized target in breast cancer that persists in many patients after HER2 targeted treatment relapse; a novel MOA to this target may impart additional clinical benefit. The potency, reduced immunogenicity, unique mechanism of action and immune modulating activities of ETBs in this second generation scaffold allows for these agents to fit in well in a refractory setting as well as in combination with other agents in the growing field of immuno-oncology. Citation Format: Sangeetha Rajagopalan, Garrett L. Robinson, Brigitte Brieschke, Jennifer Erdman, Jane Neill, Jack P. Higgins, Erin K. Willert. Next-generation engineered toxin bodies: CD38, PD-L1 and HER2 targeted ETBs. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 595.

OTSSP167 Abrogates Mitotic Checkpoint through Inhibiting Multiple Mitotic Kinases.

OTSSP167 was recently characterized as a potent inhibitor for maternal embryonic leucine zipper kinase (MELK) and is currently tested in Phase I clinical trials for solid tumors that have not responded to other treatment. Here we report that OTSSP167 abrogates the mitotic checkpoint at concentrations used to inhibit MELK. The abrogation is not recapitulated by RNAi mediated silencing of MELK in cells. Although OTSSP167 indeed inhibits MELK, it exhibits off-target activity against Aurora B kinase in vitro and in cells. Furthermore, OTSSP167 inhibits BUB1 and Haspin kinases, reducing phosphorylation at histones H2AT120 and H3T3 and causing mislocalization of Aurora B and associated chromosomal passenger complex from the centromere/kinetochore. The results suggest that OTSSP167 may have additional mechanisms of action for cancer cell killing and caution the use of OTSSP167 as a MELK specific kinase inhibitor in biochemical and cellular assays.

Differential efficacy of the TSPO ligands etifoxine and XBD-173 in two rodent models of Multiple Sclerosis

Neurosteroids such as progesterone and allopregnanolone have been shown to exert neuroprotective effects under a variety of pathological or insult conditions, and there is evidence that the neurosteroid system is perturbed in Multiple Sclerosis (MS) patients. Neurosteroids are synthesized in the central nervous system (CNS) through a series of metabolic transformations, beginning with a rate-limiting step of cholesterol transport through the outer mitochondrial membrane via the transporter translocator protein (TSPO). We examined the effects of etifoxine and XBD-173, two different brain penetrant TSPO agonists, for their ability to ameliorate clinical signs in two different experimental autoimmune encephalitis (EAE) models. Etifoxine, as previously reported, was efficacious in EAE, while XBD-173 was not. Surprisingly, XBD-173, but not etifoxine elevated relevant neurosteroids in brain of female rats and differed in its ability to exert anti-inflammatory and direct neuroprotective effects in vitro as compared to etifoxine. We conclude that the neurosteroid elevations produced in brain by XBD-173 are not sufficient to ameliorate EAE and suggest that etifoxine may have additional mechanisms of action that provide therapeutic benefit in this model system.

Glucagon-like peptide-1 improves beta-cell antioxidant capacity via extracellular regulated kinases pathway and Nrf2 translocation

Oxidative stress plays an important role in the development of beta-cell dysfunction and insulin resistance, two major pathophysiological abnormalities of type 2 diabetes. Expression levels of antioxidant enzymes in beta cells are very low, rendering them more susceptible to damage caused by reactive oxygen species (ROS). Although the antioxidant effects of glucagon-like peptide-1 (GLP-1) and its analogs have been previously reported, the exact mechanisms involved are still unclear. In this study, we demonstrated that GLP-1 was able to effectively inhibit oxidative stress and cell death of INS-1E beta cells induced by the pro-oxidant tert-butyl hydroperoxide (tert-BOOH). Incubation with GLP-1 enhanced cellular levels of glutathione and the activity of its related enzymes, glutathione-peroxidase (GPx) and -reductase (GR) in beta cells. However, inhibition of ERK, but not of the PI3K/AKT pathway abolished, at least in part, the antioxidant effect of GLP-1. Moreover, ERK activation seems to be protein kinase A (PKA)-dependent because inhibition of PKA with H-89 was sufficient to block the GLP-1-derived protective effect on beta cells. GLP-1 likewise increased the synthesis of GR and favored the translocation of the nuclear transcription factor erythroid 2p45-related factor (Nrf2), a transcription factor implicated in the expression of several antioxidant/detoxificant enzymes. Glucose-stimulated insulin secretion was also preserved in beta-cells challenged with tert-BOOH but pre-treated with GLP-1, probably through the down-regulation of the mitochondrial uncoupling-protein2 (UCP2). Thus, our results provide additional mechanisms of action of GLP-1 to prevent oxidative damage in beta cells through the modulation of signaling pathways involved in antioxidant enzyme regulation.

Kappa Opioids, Salvinorin A and Major Depressive Disorder.

Opioids are traditionally associated with pain, analgesia and drug abuse. It is now clear, however, that the opioids are central players in mood. The implications for mood disorders, particularly clinical depression, suggest a paradigm shift from the monoamine neurotransmitters to the opioids either alone or in interaction with monoamine neurons. We have a special interest in dynorphin, the last of the major endogenous opioids to be isolated and identified. Dynorphin is derived from the Greek word for power, dynamis, which hints at the expectation that the neuropeptide held for its discoverers. Yet, dynorphin and its opioid receptor subtype, kappa, has always taken a backseat to the endogenous b-endorphin and the exogenous morphine that both bind the mu opioid receptor subtype. That may be changing as the dynorphin/ kappa system has been shown to have different, often opposite, neurophysiological and behavioral influences. This includes major depressive disorder (MDD). Here, we have undertaken a review of dynorphin/ kappa neurobiology as related to behaviors, especially MDD. Highlights include the unique features of dynorphin and kappa receptors and the special relation of a plant-based agonist of the kappa receptor salvinorin A. In addition to acting as a kappa opioid agonist, we conclude that salvinorin A has a complex pharmacologic profile, with potential additional mechanisms of action. Its unique neurophysiological effects make Salvinorina A an ideal candidate for MDD treatment research.

Novel Piperazine Arylideneimidazolones Inhibit the AcrAB-TolC Pump in Escherichia coli and Simultaneously Act as Fluorescent Membrane Probes in a Combined Real-Time Influx and Efflux Assay.

In this study, we tested five compounds belonging to a novel series of piperazine arylideneimidazolones for the ability to inhibit the AcrAB-TolC efflux pump. The biphenylmethylene derivative (BM-19) and the fluorenylmethylene derivative (BM-38) were found to possess the strongest efflux pump inhibitor (EPI) activities in the AcrAB-TolC-overproducingEscherichia colistrain 3-AG100, whereas BM-9, BM-27, and BM-36 had no activity at concentrations of up to 50 μM in a Nile red efflux assay. MIC microdilution assays demonstrated that BM-19 at 1/4 MIC (intrinsic MIC, 200 μM) was able to reduce the MICs of levofloxacin, oxacillin, linezolid, and clarithromycin 8-fold. BM-38 at 1/4 MIC (intrinsic MIC, 100 μM) was able to reduce only the MICs of oxacillin and linezolid (2-fold). Both compounds markedly reduced the MIC of rifampin (BM-19, 32-fold; and BM-38, 4-fold), which is suggestive of permeabilization of the outer membrane as an additional mechanism of action. Nitrocefin hydrolysis assays demonstrated that in addition to their EPI activity, both compounds were in fact weak permeabilizers of the outer membrane. Moreover, it was found that BM-19, BM-27, BM-36, and BM-38 acted as near-infrared-emitting fluorescent membrane probes, which allowed for their use in a combined influx and efflux assay and thus for tracking of the transport of an EPI across the outer membrane by an efflux pump in real time. The EPIs BM-38 and BM-19 displayed the most rapid influx of all compounds, whereas BM-27, which did not act as an EPI, showed the slowest influx.

A chemical-genetic interaction map of small molecules using high-throughput imaging in cancer cells.

Small molecules often affect multiple targets, elicit off-target effects, and induce genotype-specific responses. Chemical genetics, the mapping of the genotype dependence of a small molecule's effects across a broad spectrum of phenotypes can identify novel mechanisms of action. It can also reveal unanticipated effects and could thereby reduce high attrition rates of small molecule development pipelines. Here, we used high-content screening and image analysis to measure effects of 1,280 pharmacologically active compounds on complex phenotypes in isogenic cancer cell lines which harbor activating or inactivating mutations in key oncogenic signaling pathways. Using multiparametric chemical-genetic interaction analysis, we observed phenotypic gene-drug interactions for more than 193 compounds, with many affecting phenotypes other than cell growth. We created a resource termed the Pharmacogenetic Phenome Compendium (PGPC), which enables exploration of drug mode of action, detection of potential off-target effects, and the generation of hypotheses on drug combinations and synergism. For example, we demonstrate that MEK inhibitors amplify the viability effect of the clinically used anti-alcoholism drug disulfiram and show that the EGFR inhibitor tyrphostin AG555 has off-target activity on the proteasome. Taken together, this study demonstrates how combining multiparametric phenotyping in different genetic backgrounds can be used to predict additional mechanisms of action and to reposition clinically used drugs.

PAIN IN NEWBORNS. P ART II. PHARMACOLOGICAL METHODS FOR PAIN RELIEF

For the treatment of pain in patients of all age categories are widely used non-narcotic analgesics, but convincing evidence of efficacy, both non-steroidal anti-inflammatory drugs (NSAIDs) and paracetamol for the relief of pain in newborn does not exist. Antipyretic and analgesic properties of paracetamol and NSAIDs are due to inhibition of cyclooxygenase activity and the inhibition of prostaglandin synthesis. However, acetaminophen differs from NSAIDs absence of anti-inflammatory properties. An additional mechanism of action of paracetamol is the impact of its active metabolites in the endogenous cannabinoid system with a resulting decrease in anxiety, motor activity, muscle tone, decreased body temperature, decreasing symptoms of pain. In addition, the central nociceptive effect of paracetamol is associated with its effects on serotoninergic system and the influence on the transmission of nociceptive signal at the level of spinal cord. When combined with morphine, paracetamol has sparring effect. The toxic effect of paracetamol on liver cells caused by its active metabolites, which depletes the liver from glutathione, and directly damages cells in the liver, which leads to necrosis and cell death, and the subsequent development of liver failure. In newborn activity of enzymes that contribute to the formation of toxic metabolites is decreased, so they rarely develop acetamenophen-induced liver damage. It shows the relationship between the intake of paracetamol in infancy and the risk of asthma or other atopic diseases. The use of narcotic analgesics may be accompanied by respiratory depression, impaired hemodynamic, inhibition of intestinal motility. As the long-term effects of pain in the neonatal period the altered sleep-wake cycles, the development of hyperalgesia and hypersensitivity and cognitive disorders was observed. Both neonatal morphine exposure and repetitive pain have been shown to cause identical disorders: behavioral changes and abnormal learning. It is study the neuroprotective effects of dexmedetomidine, highly selective agonist of α-2 adrenergic receptors, which in combination with narcotic analgesics may minimize anesthetic-induced apoptosis and to reduce the negative influence of narcotic analgesics in the child's developing brain.

Review article: inhibition of methanogenic archaea by statins as a targeted management strategy for constipation and related disorders.

SummaryBackgroundObservational studies show a strong association between delayed intestinal transit and the production of methane. Experimental data suggest a direct inhibitory activity of methane on the colonic and ileal smooth muscle and a possible role for methane as a gasotransmitter. Archaea are the only confirmed biological sources of methane in nature and Methanobrevibacter smithii is the predominant methanogen in the human intestine.AimTo review the biosynthesis and composition of archaeal cell membranes, archaeal methanogenesis and the mechanism of action of statins in this context.MethodsNarrative review of the literature.ResultsStatins can inhibit archaeal cell membrane biosynthesis without affecting bacterial numbers as demonstrated in livestock and humans. This opens the possibility of a therapeutic intervention that targets a specific aetiological factor of constipation while protecting the intestinal microbiome. While it is generally believed that statins inhibit methane production via their effect on cell membrane biosynthesis, mediated by inhibition of the HMG‐CoA reductase, there is accumulating evidence for an alternative or additional mechanism of action where statins inhibit methanogenesis directly. It appears that this other mechanism may predominate when the lactone form of statins, particularly lovastatin lactone, is administered.ConclusionsClinical development appears promising. A phase 2 clinical trial is currently in progress that evaluates the effect of lovastatin lactone on methanogenesis and symptoms in patients with irritable bowel syndrome with constipation. The review concludes with an outlook for the future and subsequent work that needs to be done.

Sotagliflozin as a potential treatment for type 2 diabetes mellitus

Introduction: SGLT1 is the primary transporter responsible for the absorption of glucose and galactose in the intestine, while SGLT2 and SGLT1 are both involved in the renal reabsorption of glucose. SGLT2 inhibitors are a new class of oral antidiabetic drugs, acting by increasing urinary glucose excretion (UGE). They offer the advantages of a reduced risk of hypoglycaemia, a decrease in body weight and blood pressure and an efficacy at all stages of type 2 diabetes (T2DM).Areas covered: Herein, the authors focus specifically on sotagliflozin (LX4211), the first-in-class dual SGLT1/SGLT2 inhibitor. Original publications in English were selected as the basis of this review. Clinical trials were identified using the Clinicaltrial.gov database.Expert opinion: By a potential additional mechanism of action on intestinal glucose absorption linked to SGLT1 inhibition, sotagliflozin differentiates from SGLT2 inhibitors by reducing postprandial glucose excursion and insulin secretion, as well as by increasing GLP-1 secretion. Despite a weaker effect on UGE than selective SGLT2 inhibitors, sotagliflozin is as effective as SGLT2 inhibitors on HbA1C reduction, with a similar safety profile in short-term studies. While sotagliflozin was first assessed in T2DM, it is now in phase 3 development as an adjuvant treatment in patients with T1DM after positive results from a pilot study.

The beneficial effects of berries on cognition, motor behaviour and neuronal function in ageing.

Previously, it has been shown that strawberry (SB) or blueberry (BB) supplementations, when fed to rats from 19 to 21 months of age, reverse age-related decrements in motor and cognitive performance. We have postulated that these effects may be the result of a number of positive benefits of the berry polyphenols, including decreased stress signalling, increased neurogenesis, and increased signals involved in learning and memory. Thus, the present study was carried out to examine these mechanisms in aged animals by administering a control, 2 % SB- or 2 % BB-supplemented diet to aged Fischer 344 rats for 8 weeks to ascertain their effectiveness in reversing age-related deficits in behavioural and neuronal function. The results showed that rats consuming the berry diets exhibited enhanced motor performance and improved cognition, specifically working memory. In addition, the rats supplemented with BB and SB diets showed increased hippocampal neurogenesis and expression of insulin-like growth factor 1, although the improvements in working memory performance could not solely be explained by these increases. The diverse polyphenolics in these berry fruits may have additional mechanisms of action that could account for their relative differences in efficacy.

Metformin Treatment for the Prevention and/or Treatment of Breast/Mammary Tumorigenesis.

There is increasing interest in metformin's effects on the development, treatment and/or progression of breast cancer. This emerges from observational studies that diabetic women treated with metformin in comparison to other antidiabetic compounds had lower breast cancer incidence and/or mortality rates. The mechanism of action is considered to be activation of hepatic AMPK resulting in reduced gluconeogenesis. Calorie restriction, which consistently reduces mammary tumorigenesis in rodents, is also thought to act through this pathway leading to the hypothesis that metformin's anticancer effects are mediated in a similar fashion. Here we review the literature evaluating metformin's anticancer effects in relation to breast/mammary tumorigenesis. We include clinical observations, as well as studies utilizing rodent models and mammary cell lines. In addition to the anticancer effect of metformin mediated through the AMPK pathway, additional mechanisms of action that directly target tissues have been identified including effects on stem cells, apoptosis, STAT3 and HER2.

Stabilization of nontoxic Aβ-oligomers: insights into the mechanism of action of hydroxyquinolines in Alzheimer's disease.

The extracellular accumulation of amyloid β (Aβ) peptides is characteristic of Alzheimer's disease (AD). However, formation of diffusible, oligomeric forms of Aβ, both on and off pathways to amyloid fibrils, is thought to include neurotoxic species responsible for synaptic loss and neurodegeneration, rather than polymeric amyloid aggregates. The 8-hydroxyquinolines (8-HQ) clioquinol (CQ) and PBT2 were developed for their ability to inhibit metal-mediated generation of reactive oxygen species from Aβ:Cu complexes and have both undergone preclinical and Phase II clinical development for the treatment of AD. Their respective modes of action are not fully understood and may include both inhibition of Aβ fibrillar polymerization and direct depolymerization of existing Aβ fibrils. In the present study, we find that CQ and PBT2 can interact directly with Aβ and affect its propensity to aggregate. Using a combination of biophysical techniques, we demonstrate that, in the presence of these 8-HQs and in the absence of metal ions, Aβ associates with two 8-HQ molecules and forms a dimer. Furthermore, 8-HQ bind Aβ with an affinity of 1-10 μm and suppress the formation of large (>30 kDa) oligomers. The stabilized low molecular weight species are nontoxic. Treatment with 8-HQs also reduces the levels of in vivo soluble oligomers in a Caenorhabditis elegans model of Aβ toxicity. We propose that 8-HQs possess an additional mechanism of action that neutralizes neurotoxic Aβ oligomer formation through stabilization of small (dimeric) nontoxic Aβ conformers.

Pharmacotherapy and analysis of gaseous mediators in hypertensive patients

To evaluate the effect of using antihypertensive classes of drugs of the calcium channel antagonists and inhibitors of angiotensin-converting enzyme in plasma concentrations of hydrogen sulfide and nitric oxide in patients with hypertension. Cross-sectional study with quantitative approach conducted with hypertensive patients in use of antihypertensive classes of drugs: angiotensin-converting enzyme inhibitors or calcium channel antagonists. It was found that the concentration of plasma nitric oxide was significantly higher in hypertensive patients that were in use of angiotensin-converting enzyme inhibitors (p<0.03) and the hydrogen sulphide concentration was significantly higher in hypertensive plasma in use of calcium channel antagonists (p<0.002). The findings suggest that these medications have as additional action mechanism the improvement of endothelial dysfunction by elevate plasma levels of vasodilatory substances.

In vivo profiling reveals immunomodulatory effects of sorafenib and dacarbazine on melanoma

Sorafenib is a multi-kinase inhibitor used alone or in combination with dacarbazine to treat metastasized melanoma. Our study investigated the relationship between metabolic response assessed by PET-CT and global transcriptome changes during sorafenib and dacarbazine therapy in patients with advanced melanoma. We conducted an open-label, investigator-initiated study that enrolled 13 sorafenib-naïve Stage IV melanoma patients, whose metastases were accessible for repeated biopsies. Treatment regimen included orally administered sorafenib and intravenous dacarbazine. Biopsies of skin or superficial lymph node metastases were taken before treatment (baseline), during sorafenib and after dacarbazine therapy and used for transcriptional profiling and validation experiments. Serum samples were evaluated for cytokine production. Metabolic response to therapy was observed in 45.5% of patients. The study drugs were well tolerated. We observed a clear upregulation of interferon (IFN)-stimulated immune response genes in profiled metastases. The IFNγ-induced gene signature seemed to be enhanced after addition of dacarbazine to sorafenib. Serum IFNγ also increased during therapy, particularly after addition of dacarbazine. Induction of IFNγ stimulated genes correlating with increased serum IFNγ was predictive of better clinical outcome and responders who had significantly higher serum IFNγ levels lived longer. Our data reveal in situ changes in melanoma metastases during treatment with sorafenib and dacarbazine and suggest an additional mechanism of action through immunomodulation.

Tamoxifen Directly Disrupts Platelet Angiogenic Potential and Inhibits Platelet-Mediated Metastasis

Platelets, which are mainly known for their role in hemostasis, are now known to play a crucial role in metastasis. Metastatic disease is the cause of roughly 90% of all cancer-related deaths and understanding the mechanisms leading to dissemination of tumor cells to distant sites remains one of the main challenges of cancer research. Tamoxifen is a selective estrogen receptor modulator that is widely used for the treatment and prevention of breast cancer. Interestingly, tamoxifen has demonstrated anti-cancer efficacy in estrogen negative breast cancers suggesting that this drug has additional mechanisms of action. Previously tamoxifen and its metabolites have been shown directly impact platelet function; however, the reported effects have been varied (Vitseva et al,. 2005, Nayak et al., 2011). Because platelets are critical for metastatic spread, we posited that tamoxifen may exert anti-tumor effects by directly altering platelet function. To explore this, we first examined the effect of tamoxifen on platelet activation. P-selectin expression was determined by flow cytometry for platelets pretreated with or without tamoxifen and then activated with ADP or MCF-7 cells. Both ADP and exposure to MCF-7 cells resulted in platelet activation and treatment with tamoxifen lead to a partial inhibition of activation. Platelets are a reservoir for angiogenic proteins that are secreted in a differentially regulated process. We have previously shown that we can manipulate the angiogenic potential of the platelet releasate through physiological (platelet agonists) and pathological activation (MCF-7 tumor cells) (Battinelli et al., 2011). We hypothesized that tamoxifen may impact malignancy by altering the release of angiogenesis regulatory proteins from platelets. To explore the impact of tamoxifen on the angiogenic potential of platelets, we analyzed the releasates from platelets exposed to tamoxifen alone or activated with ADP or MCF-7 cells in conjunction with tamoxifen. Our data reveals that platelets exposed to tamoxifen release significantly decreased amounts of VEGF in response to activation by either the platelet agonist ADP or interaction with tumor cells (MCF-7 cells). Next, in vitro angiogenesis assays were performed to further examine the effect of tamoxifen on the angiogenic potential of platelets. We observed dramatically diminished capillary tube branch point formation and decreased migration in endothelial cell cultures exposed to releasates generated from tamoxifen treated platelets compared to control releasates, demonstrating that tamoxifen inhibits the ability of activated platelets to promote angiogenesis. Platelets play a critical role in aiding the intravasation and extravasation of tumor cells in the circulation and therefore we postulated that tamoxifen could alter the ability of platelets to aid tumor cells in crossing the vascular endothelium. We performed transendothelial migration assays in which platelets were pretreated with tamoxifen or vehicle control, washed and mixed with MCF-7 tumor cells in endothelialized transwells. Platelets significantly increased the number of tumor cells that crossed the endothelial barrier; however this increase was lost when platelets were pretreated with tamoxifen. Overall our results demonstrate that tamoxifen directly alters platelet function, leading to decreased angiogenic and metastatic potential. These studies highlight a previously unknown mechanism of action for tamoxifen and may shed light on the efficacy of tamoxifen in estrogen-receptor negative cancers. Furthermore our work stresses the importance of crosstalk between platelets and cells within the tumor microenvironment for successful angiogenesis and metastatic spread and, ultimately, support the idea of utilizing targeted platelet therapies to inhibit the platelet’s role in malignancy. DisclosuresNo relevant conflicts of interest to declare.

Novel TCR-like Bi-Specific T Cell Engaging Antibody Targeting the PR1/HLA-A2 Myeloid Leukemia Antigen

PR1 is a HLA-A2 restricted immune-dominant peptide derived from proteinase 3 and neutrophil elastase - leukemia associated self-antigens aberrantly over-expressed in myeloid leukemia. The persistent PR1/HLA-A2 specific cytotoxic T cell responses are associated with prolonged remission from myeloid leukemia, suggesting that PR1/HLA-A2 would be a potent target for T cell based immunotherapy. Previously, we have successfully developed a high affinity T cell receptor (TCR)-like antibody specific for the PR1/HLA-A2 complex (h8F4), and demonstrated that h8F4 mediated specific lyses of myeloid leukemia cells invitro via antibody dependent cell mediated cytotoxicity (ADCC), and significantly reduced leukemic blasts invivo in murine xenograft model of treatment refractory acute myeloid leukemia. We hypothesize that the therapeutic efficacy of h8F4 would be enhanced by combining T cell mediated cytotoxicity through a novel antibody engineering technology, called, Bi-Specific T cell Engager (BiTE) specific for T cells and tumor associated antigens. The BiTE molecules redirect effector function of polyclonal T cells against the tumor leading to lyses of tumor cells, and early clinical data show promising therapeutic efficacy of this approach. In the current study, we constructed Bi-Specific T cell Engaging antibody: h8F4-BiTE, a single chain Fragment of variable domain (scFv) of h8F4 conjugated with scFv of anti-CD3 antibody – OKT3, and produced h8F4-BiTE proteins using eukaryotic expression system. The h8F4-BiTE proteins retained the similar binding specificity to PR1/HLA-A2 as compared to the parent antibody (h8F4), demonstrated by flowcytometry based binding assays with a series of alanine substituted PR1 peptide/HLA-A2 complexes on T2 cells. The 8F4-BiTE bound to native and endogenous PR1/HLA-A2 complexes presented by HLA-A2 positive or transduced myeloid cell lines such as K562, THP-1, and HL-60. In addition, h8F4-BiTE showed comparable binding affinity to CD3 moiety of T cells. Lastly, h8F4-BiTE proteins activated T cells in a PR1/HLA-A2 specific and dose dependent manner, shown by increased expression of activation markers such as CD69 when co-cultured with PR1-pulsed T2 target cells in various concentrations of h8F4-BiTE proteins. Importantly, the PR1/HLA-A2 specific activation of T cells by h8F4-BiTE proteins was equally efficient among all subsets of T cells regardless of their phenotypes, e.g., CD4 vs CD8, and naïve vs memory, suggesting that h8F4-BiTE would be a highly potent inducer of cytotoxic T cells against PR1/HLA-A2 positive myeloid leukemia. Moreover, we generated multiple h8F4-BiTE variants with specific mutations in frameworks and/or complement determining regions (CDRs), and compared binding affinity and function to wildtype h8F4-BiTE. The h8F4-BiTE variants indeed improved specificity to PR1/HLA-A2 complex by complete elimination of residual non-specific binding to HLA-A2, and increased the binding affinity to CD3 by 10-folds resulting in dramatic 50 fold increase in potency to activate T cells. In conclusion, we have developed a novel immunetherapeutics: h8F4-BiTE targeting PR1/HLA-A2 myeloid leukemia antigen. We demonstrated high binding affinity and specificity of h8F4-BiTE to both PR1/HLA-A2 on myeloid leukemia cells and CD3 on T cells and in vitro activation of T cells in the presence of PR1/HLA-A2 expressing target cells in a dose dependent manner. Finally, we improved antigen specificity and functional activity of h8F4-BiTE via selective site-directed mutagenesis. Further preclinical evaluation of therapeutic efficacy is thus warranted using animal in vivo animal treatment model with NOD/SCID/IL2Rγ −/− mice xenografted with human myeloid leukemia cells. This unique TCR-like therapeutic agent adds an additional mechanism of action to PR1/HLA-A2 specific antibody (h8F4) and promises to enhance T cell function against myeloid leukemia. DisclosuresNo relevant conflicts of interest to declare.

Breast cancer anti-estrogen resistance 3 inhibits transforming growth factor β/Smad signaling and associates with favorable breast cancer disease outcomes.

IntroductionThis study helps to define the implications of breast cancer anti-estrogen resistance 3 (BCAR3) in breast cancer and extends the current understanding of its molecular mechanism of action. BCAR3 has been shown to promote cell proliferation, migration and attachment to extracellular matrix components. However, in a cohort of metastatic breast cancer patients who received tamoxifen treatment, high BCAR3 mRNA levels were associated with favorable progression-free survival outcome. These results suggest that, besides its established roles, BCAR3 may have additional mechanisms of action that regulate breast cancer aggressive phenotype. In this study, we investigated whether BCAR3 is a novel antagonist of the canonical transforming growth factor β (TGFβ) pathway, which induces potent migration and invasion responses in breast cancer cells.MethodsWe surveyed functional genomics databases for correlations between BCAR3 expression and disease outcomes of breast cancer patients. We also studied how BCAR3 could regulate the TGFβ/Smad signaling axis using Western blot analysis, coimmunoprecipitation and luciferase assays. In addition, we examined whether BCAR3 could modulate TGFβ-induced cell migration and invasion by using an automated imaging system and a confocal microscopy imaging–based matrix degradation assay, respectively.ResultsRelatively low levels of BCAR3 expression in primary breast tumors correlate with poor distant metastasis-free survival and relapse-free survival outcomes. We also found a strong correlation between the loss of heterozygosity at BCAR3 gene alleles and lymph node invasion in human breast cancer, further suggesting a role for BCAR3 in preventing disease progression. In addition, we found BCAR3 to inhibit Smad activation, Smad-mediated gene transcription, Smad-dependent cell migration and matrix digestion in breast cancer cells. Furthermore, we found BCAR3 to be downregulated by TGFβ through proteasome degradation, thus defining a novel positive feedback loop mechanism downstream of the TGFβ/Smad signaling pathway.ConclusionBCAR3 is considered to be associated with aggressive breast cancer phenotypes. However, our results indicate that BCAR3 acts as a putative suppressor of breast cancer progression by inhibiting the prometastatic TGFβ/Smad signaling pathway in invasive breast tumors. These data provide new insights into BCAR3’s molecular mechanism of action and highlight BCAR3 as a novel TGFβ/Smad antagonist in breast cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-014-0476-9) contains supplementary material, which is available to authorized users.

Antiplasmodial activity of chloroquine analogs against chloroquine-resistant parasites, docking studies and mechanisms of drug action.

BackgroundGiven the threat of resistance of human malaria parasites, including to artemisinin derivatives, new agents are needed. Chloroquine (CQ) has been the most widely used anti-malarial, and new analogs (CQAns) presenting alkynes and side chain variations with high antiplasmodial activity were evaluated.MethodsSix diaminealkyne and diaminedialkyne CQAns were evaluated against CQ-resistant (CQ-R) (W2) and CQ-sensitive (CQ-S) (3D7) Plasmodium falciparum parasites in culture. Drug cytotoxicity to a human hepatoma cell line (HepG2) evaluated, allowed to calculate the drug selectivity index (SI), a ratio of drug toxicity to activity in vitro. The CQAns were re-evaluated against CQ-resistant and -sensitive P. berghei parasites in mice using the suppressive test. Docking studies with the CQAns and the human (HssLDH) or plasmodial lactate dehydrogenase (PfLDH) enzymes, and, a β-haematin formation assay were performed using a lipid as a catalyst to promote crystallization in vitro.ResultsAll tested CQAns were highly active against CQ-R P. falciparum parasites, exhibiting half-maximal inhibitory concentration (IC50) values below 1 μΜ. CQAn33 and CQAn37 had the highest SIs. Docking studies revealed the best conformation of CQAn33 inside the binding pocket of PfLDH; specificity between the residues involved in H-bonds of the PfLDH with CQAn37. CQAn33 and CQAn37 were also shown to be weak inhibitors of PfLDH. CQAn33 and CQAn37 inhibited β-haematin formation with either a similar or a 2-fold higher IC50 value, respectively, compared with CQ. CQAn37 was active in mice with P. berghei, reducing parasitaemia by 100%. CQAn33, -39 and -45 also inhibited CQ-resistant P. berghei parasites in mice, whereas high doses of CQ were inactive.ConclusionsThe presence of an alkyne group and the size of the side chain affected anti-P. falciparum activity in vitro. Docking studies suggested a mechanism of action other than PfLDH inhibition. The β-haematin assay suggested the presence of an additional mechanism of action of CQAn33 and CQAn37. Tests with CQAn34, CQAn37, CQAn39 and CQAn45 confirmed previous results against P. berghei malaria in mice, and CQAn33, 39 and 45 were active against CQ-resistant parasites, but CQAn28 and CQAn34 were not. The result likely reflects structure-activity relationships related to the resistant phenotype.