Capturing the Effects of Single Atom Substitutions on the Inhibition Efficiency of Glycogen Synthase Kinase-3ß Inhibitors via Markov State Modeling and Experiments.

Small modifications in the chemical structure of ligands are known to dramatically change their ability to inhibit the activity of a protein. Unraveling the mechanisms that govern these dramatic changes requires scrutinizing the dynamics of protein-ligand binding and unbinding at the atomic level. As an exemplary case, we have studied Glycogen Synthase Kinase-3ß (GSK-3ß), a multifunctional kinase that has been implicated in a host of pathological processes. As such, there is a keen interest in identifying ligands that inhibit GSK-3ß activity. One family of compounds that are highly selective and potent inhibitors of GSK-3ß is exemplified by a molecule termed COB-187. COB-187 consists of a five-member heterocyclic ring with a thione at C2, a pyridine substituted methyl at N3, and a hydroxyl and phenyl at C4. We have studied the inhibition of GSK-3ß by COB-187-related ligands that differ in a single heavy atom from each other (either in the location of nitrogen in their pyridine ring, or with the pyridine ring replaced by a phenyl ring), or in the length of the alkyl group joining the pyridine and the N3. The inhibition experiments show a large range of half-maximal inhibitory concentration (IC50) values from 10 nM to 10 µM, implying that these ligands exhibit vastly different propensities to inhibit GSK-3ß. To explain these differences, we perform Markov State Modeling (MSM) using fully atomistic simulations. Our MSM results are in excellent agreement with the experiments in that they accurately capture differences in the binding propensities of the ligands. The simulations show that the binding propensities are related to the ligands' ability to attain a compact conformation where their two aromatic rings are spatially close. We rationalize this result by sampling numerous binding and unbinding events via funnel metadynamics simulations, which show that indeed while approaching the bound state, the ligands prefer to be in their compact conformation. We find that the presence of nitrogen in the aromatic ring increases the probability of attaining the compact conformation. Protein-ligand binding is understood to be dictated by the energetics of interactions and entropic factors, like the release of bound water from the binding pockets. This work shows that changes in the conformational distribution of ligands due to atom-level modifications in the structure play an important role in protein-ligand binding.

Mechanistic insights into SARS-CoV-2 spike protein induction of the chemokine CXCL10.

During a SARS-CoV-2 infection, macrophages recognize viral components resulting in cytokine production. While this response fuels virus elimination, overexpression of cytokines can lead to severe COVID-19. Previous studies suggest that the spike protein (S) of SARS-CoV-2 can elicit cytokine production via the transcription factor NF-κB and the toll-like receptors (TLRs). In this study, we found that: (i) S and the S2 subunit induce CXCL10, a chemokine implicated in severe COVID-19, gene expression by human macrophage cells (THP-1); (ii) a glycogen synthase kinase-3 inhibitor attenuates this induction; (iii) S and S2 do not activate NF-κB but do activate the transcription factor IRF; (iv) S and S2 do not require TLR2 to elicit CXCL10 production or activate IRF; and (v) S and S2 elicit CXCL10 production by peripheral blood mononuclear cells (PBMCs). We also discovered that the cellular response, or lack thereof, to S and S2 is a function of the recombinant S and S2 used. While such a finding raises the possibility of confounding LPS contamination, we offer evidence that potential contaminating LPS does not underly induced increases in CXCL10. Combined, these results provide insights into the complex immune response to SARS-CoV-2 and suggest possible therapeutic targets for severe COVID-19.

Anti-Inflammatory and Therapeutic Effects of a Novel Small-Molecule Inhibitor of Inflammation in a Male C57BL/6J Mouse Model of Obesity-Induced NAFLD/MAFLD.

Purpose: Non-alcoholic fatty liver disease (NAFLD), recently renamed metabolic (dysfunction) associated fatty liver disease (MAFLD), is the most common chronic liver disease in the United States. Presently, there is an intense and ongoing effort to identify and develop novel therapeutics for this disease. In this study, we explored the anti-inflammatory activity of a new compound, termed IOI-214, and its therapeutic potential to ameliorate NAFLD/MAFLD in male C57BL/6J mice fed a high fat (HF) diet. Methods: Murine macrophages and hepatocytes in culture were treated with lipopolysaccharide (LPS) ± IOI-214 or DMSO (vehicle), and RT-qPCR analyses of inflammatory cytokine gene expression were used to assess IOI-214's anti-inflammatory properties in vitro. Male C57BL/6J mice were also placed on a HF diet and treated once daily with IOI-214 or DMSO for 16 weeks. Tissues were collected and analyzed to determine the effects of IOI-214 on HF diet-induced NAFL D/MAFLD. Measurements such as weight, blood glucose, serum cholesterol, liver/serum triglyceride, insulin, and glucose tolerance tests, ELISAs, metabolomics, Western blots, histology, gut microbiome, and serum LPS binding protein analyses were conducted. Results: IOI-214 inhibited LPS-induced inflammation in macrophages and hepatocytes in culture and abrogated HF diet-induced mesenteric fat accumulation, hepatic inflammation and steatosis/hepatocellular ballooning, as well as fasting hyperglycemia without affecting insulin resistance or fasting insulin, cholesterol or TG levels despite overall obesity in vivo in male C57BL/6J mice. IOI-214 also decreased systemic inflammation in vivo and improved gut microbiota dysbiosis and leaky gut. Conclusion: Combined, these data indicate that IOI-214 works at multiple levels in parallel to inhibit the inflammation that drives HF diet-induced NAFLD/MAFLD, suggesting that it may have therapeutic potential for NAFLD/MAFLD.

Changes in Environment and Management Practices Improve Foot Health in Zoo-Housed Flamingos.

Foot lesions are a highly prevalent phenomenon among zoo-housed flamingos, with up to 99.8% of birds affected. These lesions are a recognized welfare concern, increasing the likelihood of bacterial infections, and even septicemia. Although several risk factors have been linked to foot lesions in flamingos (including age, climate, and substrate), there have been few studies looking at changes in foot lesions over time. This study tracked changes in foot lesions for an individual flock of Chilean Flamingos (97 birds) at Dublin Zoo, Ireland, over an 18-month period in response to a mandatory indoor housing order imposed by the Irish Government as a seasonal precautionary measure to prevent the spread of avian influenza. Using a pre-defined scoring system for four common types of foot lesions (hyperkeratosis, fissures, nodular lesions, and papillomatous growths), we show that providing unrestricted access to outdoor habitats and natural substrates (both terrestrial and aquatic) can improve the health and wellbeing of zoo-housed flamingos. This longitudinal study highlights the importance of regular foot health monitoring in flamingos, and the importance of natural aquatic substrates when managing flamingos. As many zoo-housed birds have been spending more time indoors on artificial substrates over recent years due to avian influenza housing orders, it is critical that we assess the impact of such changes in management and habitat access on bird health and welfare.

Evidence for investigating GSK-3 inhibitors as potential therapeutics for severe COVID-19.

A key component of severe COVID-19 is a "cytokine storm" i.e., the excessive expression of unneeded cytokines. Previous studies suggest that SARS-CoV-2 proteins can induce macrophages to secrete pro-inflammatory cytokines; a process that may involve Toll-like receptors (TLRs). Glycogen synthase kinase-3 (GSK-3) has been implicated in TLR signal transduction and a selective GSK-3 inhibitor, termed COB-187, dramatically attenuates cytokine expression induced by the TLR ligand lipopolysaccharide (LPS). In the present study, we provide evidence that the SARS-CoV-2 spike protein (S) and the S2 subunit (S2) induce production of CXCL10 (a chemokine elevated in severe COVID-19) by a human macrophage cell line. Further, we report that two clinically relevant GSK-3 inhibitors and COB-187 attenuate S and S2 protein-induced CXCL10 production. Combined, our observations provide impetus for investigating GSK-3 inhibitors as potential therapeutics for severe COVID-19.

Toll-Like Receptor 3 Is Critical to the Pancreatic Islet Milieu That Is Required for Coxsackievirus B4-Induced Type 1 Diabetes in Female Nonobese Diabetic Mice.

OBJECTIVE: Genetic and environmental influences play a role as triggers of type 1 diabetes mellitus (T1DM). Female nonobese diabetic (NOD) mice are useful for studying T1DM as they spontaneously develop T1DM, which can be accelerated by some viruses. Toll-like receptor 3 (TLR3) is believed to play a critical role in viral-induced T1DM and ß-cell destruction, because female Tlr3 knockout (Tlr3-/-) NOD mice are protected from Coxsackievirus B4 (CVB4)-induced acceleration of T1DM. However, the exact role(s) TLR3 plays in the pathogenesis of CVB4-induced T1DM remain unknown. METHODS: This longitudinal study used immunostaining, laser capture microdissection, and reverse transcription real-time polymerase chain reaction of islets from female uninfected and CVB4-infected Tlr3+/+ and Tlr3-/- NOD mice. RESULTS: Islets isolated from female Tlr3+/+ NOD mice 4 to 8 weeks of age had higher amounts of insulitis, Cxcl10, Il1b, Tnfa, and Tgfb1 expression compared with Tlr3-/- NOD mice. After CVB4 infection, Tlr3+/+ NOD mice had higher amounts of insulitis and T-cell infiltration at 3 days after infection compared with Tlr3-/- CVB4-infected NOD mice. CONCLUSIONS: Toll-like receptor 3 is necessary for establishment of a pancreatic islet inflammatory microenvironment by increasing insulitis and cytokine expression that facilitates CVB4-induced T1DM in female NOD mice.

Consequences of Both Coxsackievirus B4 and Type 1 Diabetes on Female Non-Obese Diabetic Mouse Kidneys.

Despite the 2019 Executive Order on Advancing American Kidney Health Initiative, kidney disease has moved up in rank from the 9th to the 8th leading cause of death in the United States. A recent push in the field of nephrology has been to identify molecular markers and/or molecular profiles involved in kidney disease process or injury that can help identify the cause of injury and predict patient outcomes. While these studies have had moderate success, they have not yet considered that many of the health conditions that cause kidney disease (diabetes, hypertension, etc.) can also be caused by environmental factors (such as viruses), which in and of themselves can cause kidney disease. Thus, the goal of this study was to identify molecular and phenotypic profiles that can differentiate kidney injury caused by diabetes (a health condition resulting in kidney disease) and coxsackievirus B4 (CVB4) exposure (which can cause diabetes and/or kidney disease), both alone and together. Non-obese diabetic (NOD) mice were used for this study due to their susceptibility to both type 1 diabetes (T1D)- and CVB4-mediated kidney injury, in order to glean a better understanding of how hyperglycemia and viral exposure, when occurring on their own and in combination, may alter the kidneys' molecular and phenotypic profiles. While no changes in kidney function were observed, molecular biomarkers of kidney injury were significantly up- and downregulated based on T1D and CVB4 exposure, both alone and together, but not in a predictable pattern. By combining individual biomarkers with function and phenotypic measurements (i.e., urinary albumin creatinine ratio, serum creatinine, kidney weight, and body weight), we were able to perform an unbiased separation of injury group based on the type of injury. This study provides evidence that unique kidney injury profiles within a kidney disease health condition are identifiable, and will help us to identify the causes of kidney injury in the future.

A novel GSK-3 inhibitor binds to GSK-3ß via a reversible, time and Cys-199-dependent mechanism.

Glycogen synthase kinase-3 (GSK-3) has been implicated in numerous pathologies making GSK-3 an attractive therapeutic target. Our group has identified a compound termed COB-187 that is a potent and selective inhibitor of GSK-3. In this study, we probed the mechanism by which COB-187 inhibits GSK-3ß. Progress curves, generated via real-time monitoring of kinase activity, indicated that COB-187 inhibition of GSK-3ß is time-dependent and subsequent jump dilution assays revealed that COB-187 binding to GSK-3ß is reversible. Further, a plot of the kinetic constant (kobs) versus COB-187 concentration suggested that, within the range of concentrations studied, COB-187 binds to GSK-3ß via an induced-fit mechanism. There is a critical cysteine residue at the entry to the active site of GSK-3ß (Cys-199). We generated a mutant version of GSK-3ß wherein Cys-199 was substituted with an alanine. This mutation caused a dramatic decrease in the activity of COB-187; specifically, an IC50 in the nM range for wild type versus >100 µM for the mutant. A screen of COB-187 against 34 kinases that contain a conserved cysteine in their active site revealed that COB-187 is highly selective for GSK-3 indicating that COB-187's inhibition of GSK-3ß via Cys-199 is specific. Combined, these findings suggest that COB-187 inhibits GSK-3ß via a specific, reversible, time and Cys-199-dependent mechanism.

Evidence that knock down of GSK-3ß in Chronic Myelogenous Leukemia cells augments IFN-γ-induced apoptosis.

The role of interferon-gamma (IFN-γ) in Chronic Myelogenous/Myeloid Leukemia (CML) and in the treatment of CML remains unclear; specifically, the effect of IFN-γ on apoptosis. There is reported interplay between IFN-γ and glycogen synthase kinase-3 (GSK-3), a kinase which has been implicated in both cell death and, conversely, cell survival. Thus, we utilized the CML-derived HAP1 cell line and a mutant HAP1 GSK-3ß knocked-down cell line (GSK-3ß 31bp) to investigate whether GSK-3 modulates IFN-γ's action on CML cells. Significantly less GSK-3ß 31bp cells, relative to HAP1 cells, were present after 48 h treatment with IFN-γ. IFN-γ treatment significantly decreased GSK-3ß 31bp substrate adhesiveness (relative to HAP1 cells); an observation often correlated with cell death. Fluorescence microscopy revealed that IFN-γ induces a modest level of apoptosis in the HAP1 cells and that IFN-γ induced apoptosis is significantly enhanced in GSK-3ß 31bp cells. Utilizing a complementary GSK-3ß knocked-down cell line (8bp) we found, via flow cytometric analysis, that IFN-γ induced apoptosis is significantly enhanced in GSK-3ß 8bp cells relative to HAP1 cells. Combined, our findings suggest that IFN-γ induces apoptosis of CML cells and that loss of GSK-3ß significantly augments IFN-γ-induced apoptosis.

Modulation of LPS-induced inflammatory cytokine production by a novel glycogen synthase kinase-3 inhibitor.

Sepsis is a serious condition that can lead to long-term organ damage and death. At the molecular level, the hallmark of sepsis is the elevated expression of a multitude of potent cytokines, i.e. a cytokine storm. For sepsis involving gram-negative bacteria, macrophages recognize lipopolysaccharide (LPS) shed from the bacteria, activating Toll-like-receptor 4 (TLR4), and triggering a cytokine storm. Glycogen synthase kinase-3 (GSK-3) is a highly active kinase that has been implicated in LPS-induced cytokine production. Thus, compounds that inhibit GSK-3 could be potential therapeutics for sepsis. Our group has recently described a novel and highly selective inhibitor of GSK-3 termed COB-187. In the present study, using THP-1 macrophages, we evaluated the ability of COB-187 to attenuate LPS-induced cytokine production. We found that COB-187 significantly reduced, at the protein and mRNA levels, cytokines induced by LPS (e.g. IL-6, TNF-α, IL-1ß, CXCL10, and IFN-ß). Further, the data suggest that the inhibition could be due, at least in part, to COB-187 reducing NF-κB (p65/p50) DNA binding activity as well as reducing IRF-3 phosphorylation at Serine 396. Thus, COB-187 appears to be a potent inhibitor of the cytokine storm induced by LPS.

Coxsackievirus B4 Exposure Results in Variable Pattern Recognition Response in the Kidneys of Female Non-Obese Diabetic Mice Before Establishment of Diabetes.

End-stage renal disease (ESRD) is described by four primary diagnoses, diabetes, hypertension, glomerulonephritis, and cystic kidney disease, all of which have viruses implicated as causative agents. Enteroviruses, such as coxsackievirus (CV), are a common genus of viruses that have been implicated in both diabetes and cystic kidney disease; however, little is known about how CVs cause kidney injury and ESRD or predispose individuals with a genetic susceptibility to type 1 diabetes (T1D) to kidney injury. This study evaluated kidney injury resulting from coxsackievirus B4 (CVB4) inoculation of non-obese diabetic (NOD) mice to glean a better understanding of how viral exposure may predispose individuals with a genetic susceptibility to T1D to kidney injury. The objectives were to assess acute and chronic kidney damage in CVB4-inoculated NOD mice without diabetes. Results indicated the presence of CVB4 RNA in the kidney for at least 14 days post-CVB4 inoculation and a coordinated pattern recognition receptor response, but the absence of an immune response or cytotoxicity. CVB4-inoculated NOD mice also had a higher propensity to develop an increase in mesangial area 17 weeks post-CVB4 inoculation. These studies identified initial gene expression changes in the kidney resulting from CVB4 exposure that may predispose to ESRD. Thus, this study provides an initial characterization of kidney injury resulting from CVB4 inoculation of mice that are genetically susceptible to developing T1D that may one day provide better therapeutic options and predictive measures for patients who are at risk for developing kidney disease from T1D.

Toll-Like Receptors Signaling in the Tumor Microenvironment.

The involvement of inflammation in cancer progression is well-established. The immune system can play both tumor-promoting and -suppressive roles, and efforts to harness the immune system to help fight tumor growth are at the forefront of research. Of particular importance is the inflammatory profile at the site of the tumor, with respect to both the leukocyte population numbers, the phenotype of these cells, as well as the contribution of the tumor cells themselves. In this regard, the pro-inflammatory effects of pattern recognition receptor expression and activation in the tumor microenvironment have emerged as a relevant issue both for therapy and to understand tumor development.Pattern recognition receptors (PRRs) were originally recognized as components of immune cells, particularly innate immune cells, as detectors of pathogens. PRR signaling in immune cells activates them, inducing robust antimicrobial responses. In particular, toll-like receptors (TLRs) constitute a family of membrane-bound PRRs which can recognize pathogen-associated molecular patterns (PAMPs) carried by bacteria, virus, and fungi. In addition, PRRs can recognize products generated by stressed cells or damaged tissues, namely damage-associated molecular patterns or DAMPS. Taking into account the role of the immune system in fighting tumors together with the presence of immune cells in the microenvironment of different types of tumors, strategies to activate immune cells via PRR ligands have been envisioned as an anticancer therapeutic approach.In the last decades, it has been determined that PRRs are present and functional on nonimmune cells and that their activation in these cells contributes to the inflammation in the tumor microenvironment. Both tumor-promoting and antitumor effects have been observed when tumor cell PRRs are activated. This argues against nonspecific activation of PRR ligands in the tumor microenvironment as a therapeutic approach. Therefore, the use of PRR ligands for anticancer therapy might benefit from strategies that specifically deliver these ligands to immune cells, thus avoiding tumor cells in some settings. This review focuses on these aspects of TLR signaling in the tumor microenvironment.

Identification of a novel selective and potent inhibitor of glycogen synthase kinase-3.

Glycogen synthase kinase-3 (GSK-3) is a multitasking protein kinase that regulates numerous critical cellular functions. Not surprisingly, elevated GSK-3 activity has been implicated in a host of diseases including pathological inflammation, diabetes, cancer, arthritis, asthma, bipolar disorder, and Alzheimer's. Therefore, reagents that inhibit GSK-3 activity provide a means to investigate the role of GSK-3 in cellular physiology and pathophysiology and could become valuable therapeutics. Finding a potent inhibitor of GSK-3 that can selectively target this kinase, among over 500 protein kinases in the human genome, is a significant challenge. Thus there remains a critical need for the identification of selective inhibitors of GSK-3. In this work, we introduce a novel small organic compound, namely COB-187, which exhibits potent and highly selective inhibition of GSK-3. Specifically, this study 1) utilized a molecular screen of 414 kinase assays, representing 404 unique kinases, to reveal that COB-187 is a highly potent and selective inhibitor of GSK-3; 2) utilized a cellular assay to reveal that COB-187 decreases the phosphorylation of canonical GSK-3 substrates indicating that COB-187 inhibits cellular GSK-3 activity; and 3) reveals that a close isomer of COB-187 is also a selective and potent inhibitor of GSK-3. Taken together, these results demonstrate that we have discovered a region of chemical design space that contains novel GSK-3 inhibitors. These inhibitors will help to elucidate the intricate function of GSK-3 and can serve as a starting point for the development of potential therapeutics for diseases that involve aberrant GSK-3 activity.

Phenylmethimazole abrogates diet-induced inflammation, glucose intolerance and NAFLD.

Nonalcoholic fatty liver disease (NAFLD) is the hepatic manifestation of both metabolic and inflammatory diseases and has become the leading chronic liver disease worldwide. High-fat (HF) diets promote an increased uptake and storage of free fatty acids (FFAs) and triglycerides (TGs) in hepatocytes, which initiates steatosis and induces lipotoxicity, inflammation and insulin resistance. Activation and signaling of Toll-like receptor 4 (TLR4) by FFAs induces inflammation evident in NAFLD and insulin resistance. Currently, there are no effective treatments to specifically target inflammation associated with this disease. We have established the efficacy of phenylmethimazole (C10) to prevent lipopolysaccharide and palmitate-induced TLR4 signaling. Because TLR4 is a key mediator in pro-inflammatory responses, it is a potential therapeutic target for NAFLD. Here, we show that treatment with C10 inhibits HF diet-induced inflammation in both liver and mesenteric adipose tissue measured by a decrease in mRNA levels of pro-inflammatory cytokines. Additionally, C10 treatment improves glucose tolerance and hepatic steatosis despite the development of obesity due to HF diet feeding. Administration of C10 after 16 weeks of HF diet feeding reversed glucose intolerance, hepatic inflammation, and improved hepatic steatosis. Thus, our findings establish C10 as a potential therapeutic for the treatment of NAFLD.

Modulation of double-stranded RNA pattern recognition receptor signaling in ovarian cancer cells promotes inflammatory queues.

Inflammation and cancer are inter-related, and both pro- and anti-tumorigenic effects are possible in different contexts, highlighting the importance of characterizing specific inflammatory pathways in distinct tumor types. Malignant cells and non-cancerous cells such as fibroblasts, infiltrating leukocytes (i.e., dendritic cells [DC], macrophages, or lymphocytes) and endothelial cells, in combination with the extracellular matrix, constitute the tumor microenvironment (TME). In the last decades, the role of the TME in cancer progression has gained increased attention and efforts directed at abrogating its deleterious effects on anti-cancer therapies have been ongoing. In this context, we investigated the potential of mouse and human ovarian cancer cells to produce inflammatory factors in response to pathogen recognition receptor (PRR) signaling, which might help to shape the biology of the TME. We determined that mouse ovarian tumors generate chemokines that are able to interact with receptors harbored by tumor-associated DCs. We also found that dsRNA triggers significant pro-inflammatory cytokine up-regulation in both human and mouse ovarian tumor cell lines, and that several PRR can simultaneously contribute to the stimulated inflammatory response displayed by these cells. Thus, dsRNA-activated PRRs may not only constitute potentially relevant drug targets for therapies aiming to prevent inflammation associated with leukocyte recruitment, or as co-adjuvants of therapeutic treatments, but also might have a role in development of nascent tumors, for example via activation of cancer cells by microbial molecules associated to pathogens, or with those appearing in circulation due to dysbiosis.

Phenylmethimazole and a thiazole derivative of phenylmethimazole inhibit IL-6 expression by triple negative breast cancer cells.

Inhibition of interleukin-6 (IL-6) holds significant promise as a therapeutic approach for triple negative breast cancer (TNBC). We previously reported that phenylmethimazole (C10) reduces IL-6 expression in several cancer cell lines. We have identified a more potent derivative of C10 termed COB-141. In the present work, we tested the hypothesis that C10 and COB-141 inhibit TNBC cell expressed IL-6 and investigated the potential for classical IL-6 pathway induced signaling within TNBC cells. A panel of TNBC cell lines (MDA-MB-231, Hs578T, MDA-MB-468) was used. Enzyme linked immunosorbent assays (ELISA) revealed that C10 and COB-141 inhibit MDA-MB-231 cell IL-6 secretion, with COB-141 being ~6.5 times more potent than C10. Therefore, the remainder of the study focused on COB-141 which inhibited IL-6 secretion, and was found, via quantitative real time polymerase chain reaction (QRT-PCR), to inhibit IL-6 mRNA in the TNBC panel. COB-141 had little, if any, effect on metabolic activity indicating that the IL-6 inhibition is not via a toxic effect. Flow cytometric analysis and QRT-PCR revealed that the TNBC cell lines do not express the IL-6 receptor (IL-6Rα). Trans-AM assays suggested that COB-141 exerts its inhibitory effect, at least in part, by reducing NF-κB (p65/p50) DNA binding. In summary, COB-141 is a potent inhibitor of TNBC cell expressed IL-6 and the inhibition does not appear to be due to non-specific toxicity. The TNBC cell lines do not have an intact classical IL-6 signaling pathway. COB-141's inhibitory effect may be due, at least in part, to reducing NF-κB (p65/p50) DNA binding.

TLR signaling inhibitor, phenylmethimazole, in combination with tamoxifen inhibits human breast cancer cell viability and migration.

Heightened co-expression and dysregulated signaling associated with Toll-like receptor 3 (TLR3) and Wnt5a is an integral component of solid tumors and hematological malignancies. Our previous findings in pancreatic cancer and melanoma suggest that inhibition of these pathways by a TLR3 signaling inhibitor, phenylmethimazole (C10), results in significantly decreased IL-6 levels, STAT3 phosphorylation, minimal cancer cell migration and reduced cancer cell growth in vitro and in vivo. In this study, we extended our earlier observations by performing studies in human breast cancer cells. We found that human MCF-7 breast cancer cells express high basal levels of TLR3 and Wnt5a RNA. C10 treatment resulted in significantly decreased TLR3 and Wnt5a expression levels. This functionally translated into significantly reduced IL-6 levels and STAT3 phosphorylation in vitro. In addition, the inhibition of this signaling cascade by C10 further resulted in decreased cell viability and migration of MCF-7 cells. Strikingly, the combination of C10 and tamoxifen, the standard of care therapy for breast cancer, further decrease cancer cell growth better than either agent alone. These data support the novel finding that inhibition of TLR3 signaling in combination with tamoxifen, may increase the effectiveness of current treatments of breast cancer.

Laser capture microdissection tailored to type 1 diabetes mellitus research.

RNA isolation from pancreatic islets poses unique challenges. Here, we present a reproducible means of obtaining high-quality RNA from juvenile rodent islets in sufficient quantities for use in ex vivo expression studies. Tissue was extracted from female non-obese diabetic (NOD) toll-like receptor 3 (TLR3)(+/+) and (TLR3)(-/-) mice in the pre-diabetic stage. Samples were frozen in liquid nitrogen, sectioned, fixed in a highly alcoholic solution, and stained with an alcoholic cresyl violet (CV) solution. Rehydration of the fixed sections was minimized. Islets were identified visually and isolated with the Leica LMD6000 laser capture microdissection (LCM) system to yield samples highly enriched in islet RNA. Real time qPCR was performed on the islet cDNA using probes for CXC chemokine ligand 10 (CXCL10), an inflammatory marker that plays a critical role in the pathogenesis of type 1 diabetes mellitus (TIDM). This method represents an improvement over currently described LCM techniques for rodent pancreatic islets and makes feasible expression studies using small amounts of starting tissue without the need for RNA pre-amplification. This has immediate implications for ongoing TIDM studies using the NOD mouse.

Absence of Activation of DNA Repair Genes and Excellent Efficacy of Phosphaplatins against Human Ovarian Cancers: Implications To Treat Resistant Cancers.

Phosphaplatins, platinum(II) and platinum(IV) complexes coordinated to a pyrophosphate moiety, exhibit excellent antitumor activities against a variety of cancers. To determine whether phosphaplatins trigger resistance to treatment by engaging DNA damage repair genes, a yeast genome-wide fitness assay was used. Treatment of yeast cells with pyrodach-2 (D2) or pyrodach-4 (D4) revealed no particular sensitivity to nucleotide excision repair, homologous recombination repair, or postreplication repair when compared with platin control compounds. Also, TNF receptor superfamily member 6 (FAS) protein was overexpressed in phosphaplatin-treated ovarian tumor cells, and platinum colocalized with FAS protein in lipid rafts. An overactivation of sphingomyelinase (ASMase) was noted in the treated cells, indicating participation of an extrinsic apoptotic mechanism due to increased ceramide release. Our results indicate that DNA is not the target of phosphaplatins and accordingly, that phosphaplatins might not cause resistance to treatment. Activation of ASMase and FAS along with the colocalization of platinum with FAS in lipid rafts support an extrinsic apoptotic signaling mechanism that is mediated by phosphaplatins.