THP-1 Cell Death Research Articles

Development and characterization of AgHNTs@SPU film loaded with letrozole as drug delivery system and its anticancer activity

Polyurethane is an excellent implant biomaterial due to its elasticity, flexibility, resistance to abrasions and chemicals, and highly hydrophobic nature. The risk of hydrophobic nature is a significant drawback for an ideal drug delivery system. To enhance the hydrophilicity and compatibility of polyurethane via surface modification, such as (1) Introducing salicylic acid into the polymer backbone can create voids for the release of drugs from the matrix. (2) Doping silver onto HNTs by a simple one-pot precipitation method can enable silver to rupture the nucleus of cancer cells and induce ROS formation. The polyurethane and its mixed matrix were cast into the film to improve the initial effect of the drug, duration, and dosing than other conventional methods. The benefits of surface modification and thin film technology resulted in degradability, cytocompatibility, and excellent anticancer activity. The in vitro release studies depict that the letrozole-loaded film has a sustained release effect for a longer period based on the swelling ratio. The MTT assay showed that the [email protected] film could cause THP-1 cell death due to silver, polyols of PU, and HNTs. Salicylic acid played a vital role in increasing the antibacterial activity of the film. Furthermore, the increased oxidative stress was seen in [email protected]/Let film due to the biomaterial's active presence. Thus, [email protected]/Let a film is a potential approach for producing an implantable anticancer drug delivery system for the future with higher tumor cell death and prolonged drug release.

Cytarabine-induced destabilization of MCL1 mRNA and protein triggers apoptosis in leukemia cells

Although cytarabine (Ara-C) is the mainstay of treatment for acute myeloid leukemia (AML), its cytotoxic mechanisms for inducing apoptosis are poorly understood. Therefore, we investigated the Ara-C-induced cell death pathway in human AML U937 cells. Ara-C-induced downregulation of MCL1 is associated with the induction of mitochondrial depolarization and apoptosis. Ara-C triggered NOX4-mediated ROS production, which in turn activated p38 MAPK but inactivated AKT. Ara-C-induced DNA damage modulates p38 MAPK activation without affecting AKT inactivation in U937 cells. Inactivated AKT promotes GSK3β-dependent CREB phosphorylation, which in turn increases NOXA transcription, thereby triggering the degradation of MCL1 protein. Activated p38 MAPK induces HuR downregulation, leading to accelerated MCL1 mRNA turnover. A similar pathway also explains the Ara-C-induced THP-1 cell death. Collectively, our data confirm that Ara-C-triggered apoptosis in the AML cell lines U937 and THP-1 is mediated through the destabilization of MCL1 mRNA and protein. Furthermore, Ara-C acts synergistically with the BCL2 inhibitor ABT-199 to induce cell death in ABT-199-resistant and parental U937 cells by inhibiting MCL1 expression.

Janerin Induces Cell Cycle Arrest at the G2/M Phase and Promotes Apoptosis Involving the MAPK Pathway in THP-1, Leukemic Cell Line

Janerin is a cytotoxic sesquiterpene lactone that has been isolated and characterized from different species of the Centaurea genus. In this study, janerin was isolated form Centaurothamnus maximus, and its cytotoxic molecular mechanism was studied in THP-1 human leukemic cells. Janerin inhibited the proliferation of THP-1 cells in a dose-dependent manner. Janerin caused the cell cycle arrest at the G2/M phase by decreasing the CDK1/Cyclin-B complex. Subsequently, we found that janerin promoted THP-1 cell death through apoptosis as indicated by flow cytometry. Moreover, apoptosis induction was confirmed by the upregulation of Bax, cleaved PARP-1, and cleaved caspase 3 and the downregulation of an anti-apoptotic Bcl-2 biomarker. In addition, immunoblotting indicated a dose dependent upregulation of P38-MAPK and ERK1/2 phosphorylation during janerin treatment. In conclusion, we have demonstrated for the first time that janerin may be capable of inducing cell cycle arrest and apoptosis through the MAPK pathway, which would be one of the mechanisms underlying its anticancer activity. As a result, janerin has the potential to be used as a therapeutic agent for leukemia.

Cellular and Immunological Response of THP-1 Cells in Response to Lipopolysaccharides and Lipoteichoic Acid Exposure

Introduction: Bacterial toxins are considered to be obscuring factors in multiple human diseases. Accidental contamination by bacterial lipopolysaccharides (LPS) and lipoteichoic acid (LTA) in biomedical and biotechnological products may lead to inflammation and associated pyrogenic reactions in humans. LPS are the most studied and clinically important pyrogen from gram-negative bacteria, while LTA is derived from the cell wall of gram-positive bacteria. Previous studies have identified that LPS and LTA induce a pyrogenic response in living systems. However, the detailed mechanisms of cellular toxicity have not yet been determined. The present study aimed to investigate LPS-induced and LTA-induced immune response activation and the ensuing effects on cellular integrity and organelle function in monocytes and monocyte-derived macrophages. Methods: With monocytes and macrophages being the first lines of defense against pathogenic invasion, THP-1 cells were selected as an in vitro model for the present study. THP-1 is a cancerous monocyte cell line and can differentiate into macrophages via stimulation with phorbolmyristic acid. Intracellular responses of monocytes and monocyte-derived macrophages following exposure to LPS and LTA were analyzed through several assays, including MTT, Neutral red, DCFH-DA, NO release, DilCI (5), Annexin V-PI FACS, Rhodamine phalloidine, Acridine Orange, and JC1. The expression of the inflammation-related genes NFκB, COX2, IL-1β, and TNF-α, along with activation of inflammatory signaling, was also assessed in monocytes following toxic interaction. Results & Conclusion: The results revealed that LPS and LTA can induce inflammatory signals at nanogram concentrations but are non-cytotoxic at low concentrations. LPS exposure had no significant toxic impacts on membrane integrity and organelle function. LPS induced neither apoptotic nor necrotic cell death of THP-1 cells at the selected concentration. However, LTA caused cell membrane damage and organelle dysfunction in a time and dose-dependent manner. LTA exposure led to oxidative stress and apoptotic cell death mediated by reactive oxygen species (ROS) and reactive nitrogen species (RNS). Both bacterial toxins augmented the expression of inflammatory genes and associated inflammatory signal transmission in THP-1 cells. The overall cellular effect was dependent on dose and time of exposure.

1187. CFTR Function Impacts Proinflammatory Cytokine Expression of Bronchial Epithelial Cells During Pseudomonas aeruginosa and Staphylococcus aureus Infections

BackgroundCystic fibrosis (CF) is a genetic disease in which opportunistic respiratory infections are common, particularly with P. aeruginosa and S. aureus. It has been suggested that dysfunction in the cystic fibrosis transmembrane conductance regulator (CFTR) could impact host immune response to these infections. The aim of this study was to determine if dysfunction in the CFTR gene could impact host cell death and the expression of proinflammatory cytokines.MethodsWe used two human bronchial epithelial (HBE) cell lines NuLi-1 and CuFi-1, which were recovered from individuals without and with CF, respectively. THP-1 cells were also differentiated into a macrophage phenotype with 50ng/mL PMA, and 10 µM CFTRinh-172 was used to impair CFTR function in these cells. Laboratory reference strains PAO1 and S. aureus 6538 were used to conduct in vitro infections. Host cell death at various MOIs was evaluated using a neutral red uptake assay, and cytokine expression was measured using ELISAs.ResultsNo differences in THP-1 cell death or cytokine expression were observed for infections with S. aureus 6538 or PAO1 with and without CFTRinh-172. No differences in cell death for infections with the HBE cells and either bacteria were noted. S. aureus 6538 induced higher levels of IL-1β when infecting CuFi-1 cells in comparison to NuLi-1 cells across all MOIs (P < 0.001) and induced higher levels of IL-6 at an MOI of 100 (P < 0.01). S. aureus 6538 also induced higher levels of IL-8 when infecting CuFi-1 cells in comparison to NuLi-1 at MOIs of 1 and 10 (P < 0.05 and P < 0.01, respectively). Meanwhile, PAO1 induced less IL-6 expression when infecting CuFI-1 cells at an MOI of 1 and 10 (P < 0.01 and P < 0.01, respectively). CuFi-1 cells infected with PAO1 had less IL-8 expression in comparison to infected NuLi-1 cells for all MOIs (P < 0.01), but no differences in IL-1β expression were observed when infecting either cell line with PAO1.ConclusionBronchial epithelial cells with and without functional CFTR appear to respond differently to infections with either P. aeruginosa or S. aureus. Further elucidating how various immune response pathways are impacted by CFTR dysfunction may lead to alternative approaches to therapy to reduce morbidity observed among CF patients.Disclosures All Authors: No reported disclosures

Resistance of THP-1 Leukemia Cells Infected with Cytomegalovirus to Anti-tumor Antibiotic Doxorubicin and Restoration of the Sensitivity by Inhibitors of the PI3K/AKT/mTOR Molecular Pathway.

Results obtained showed that infection with HCMV prevented the death of THP-1 cells treated with DOX in both active and latent forms of infection. In the presence of mTOR inhibitors (rapamycin and Torin2), the sensitivity of the infected cells to DOX was restored. Rapamycin inhibited the expression of the HCMV protein IE1-p72 and increased sensitivity to DOX. Molecular targets for the creation of new drugs for the treatment of leukemia in patients infected with HCMV were determined.

Pathogenicity differences of Salmonella enterica serovars Typhimurium, Enteritidis, and Choleraesuis-specific virulence plasmids and clinical S. Choleraesuis strains with large plasmids to the human THP-1 cell death

Salmonella is a common foodborne and zoonotic pathogen. Only a few serovars carry a virulence plasmid (pSV), which enhances the pathogenicity of the host. Here, we investigated the pathogenicity roles of the pSVs among wild-type, plasmid-less, and complemented S. Typhimurium, S. Enteritidis S. Choleraesuis in invasion, phagocytosis, and intracellular bacterial survival in human THP-1 cells and cell death patterns by flow cytometry and difference in cell death patterns between pig and human S. Choleraesuis isolates with large pSCVs. Virulence plasmid (pSTV) led to slightly increasing cellular apoptosis for S. Typhimurium; virulence plasmid (pSEV) enhanced apoptosis and necrosis significantly for S. Enteritidis; and pSCV reduced apoptosis significantly for S. Choleraesuis. After complementation, pSTV increased the intracellular survival of pSCV-less Choleraesuis and the cytotoxicity against human THP-1 cells. Using the Cytochalasin D to differentiate the invasion of S. Choleraaesuis and phagocytosis of THP-1 cells determined that pSCV were responsible for invasion and phagocytosis at 0 h and inhibited intracellular replication in THP-1 cells, and pSTV were responsible for invasion and increased intracellular survival for S. Choleraesuis in THP-1 cells. The human isolates with large pSCV induced more cellular apoptosis and necrosis than the pig isolates. In conclusion, human S. Choleraesuis isolates carrying large pSCVs were more adapted to human THP-1 cells for more cell death than pig isolates with large pSCV. The role of pSVs in invasion, phagocytosis, intracellular survival and apoptosis differed among hosted serovars.

Phenotypic Characteristics of Macrophages and Tumor Cells in Coculture

Immunosuppressive activity of a tumor is provided by constituents of its microenvironment (TME), immune cells, particularly macrophages, which, instead of protecting the organism, render cancer cells more aggressive and metastatically active. A solid tumor is a spacious structure; a naive macrophage, on its way through the tumor, encounters newer cell populations; and its functioning can be improved. In our study, we employed the method of sequential cocultivation of monocyte-like THP-1 cells with A431 human epidermoid cancer cells and explored the changes in the behavior of both cell counterparts depending on cocultivation stage, cytokine profiles, and metalloproteinase 2 and 9 (MMP) activity. We found that the first contact of the monocyte with tumor cells already increased the tumor cell proliferation and migration, release of protumorigenic cytokines, and monocyte polarization into M2 phenotype. One of the most abundant cytokines induced by the cocultivation was tumor necrosis factor, TNF-alpha, a potent inducer of apoptosis. Its release led to the massive death of THP-1 cells, so that at the other stages of cocultivation, a weakened population of macrophages could not affect the behavior of tumor cells. In conclusion, the macrophage function in TME strongly depends on its cytokine content and the data reported here elucidate one scenario of interaction between stromal and cancer cells in a real tumor.

Haemin-induced cell death in human monocytic cells is consistent with ferroptosis

BackgroundIron overload is a major issue for transfusion-dependent patients. Repeated transfusions result in the loading of large amounts of haem-derived iron on macrophages, and the haemin in turn induces cell death and the generation of reactive oxygen species (ROS) in both murine macrophages and human monocytic THP-1 cells. This haemin-induced cell death process has been shown to be iron-dependent. Thus, we hypothesized that haemin-induced THP-1 cell death is a result of ferroptosis, an iron-dependent mechanism of cell death regulation. Material and methodsHuman monocytic THP-1 cells were treated with haemin, and haemin-induced cell death and ROS generation were assessed using flow cytometry. ResultsHaemin-induced THP-1 cell death showed a necrosis pattern, and treatment with iron chelators suppressed both haemin-induced cell death and ROS generation. Treatment with ferrostatin-1, a ferroptosis inhibitor, suppressed haemin-induced cell death without affecting ROS generation, whereas erastin, a ferroptosis inducer, enhanced both haemin-induced cell death and ROS generation. DiscussionOur findings support haemin-induced cell death as an example of ferroptosis. Therefore, ferroptosis inhibitors may be useful for the treatment or prevention of transfusion iron overload.

Inhibition of Mitochondrial Respiration by Amino‐Functionalized Gold Nanoparticles in Acute Myeloid Leukemia Cells

In recent years, nanomaterials emerged as versatile tools for biomedical applications. Due to their special physicochemical characteristics, many different types of nanomaterials are under investigation for diagnostic as well as therapeutic purposes. We have previously shown the intrinsic cytotoxic activity of amino‐functionalized polystyrene nanoparticles towards acute myeloid leukemia cells. Here, we developed biocompatible gold nanoparticles with amino‐functionalization and investigated their mechanism of cellular toxicity.Acute myeloid leukemia (AML) is characterized by the proliferation and accumulation of mostly undifferentiated myeloid progenitor cells. In comparison to other leukemias, AML is a disease of the elderly with a mean age at diagnosis of 68 years. Due to severe side effects of the standard therapy, elderly AML patients often receive only palliative care, which results in profoundly reduced survival rates in older patients. Even though, a range of molecular changes in AML has already been investigated in detail, only few therapeutic agents have been approved. One possible explanation for the limited efficacy of targeted agents in AML might be the high level of genetic heterogeneity in myeloid leukemia.For a long time, glycolysis has been considered the major pathway of energy production in cancer cells, also known as the Warburg effect. Recently, however, oxidative phosphorylation emerged as a crucial metabolic pathway for the survival and proliferation of leukemia cells. The high dependence of acute myeloid leukemia cells on oxidative phosphorylation suggests the possibility to target the mitochondrial respiratory chain for antileukemic efficacy.Here, we performed high resolution respirometry using an Oroboros oxygraph and show the enhanced respiratory activity of THP‐1 leukemia cells compared to healthy peripheral mononuclear cells. Furthermore, chromogenic complex activity assays employed to study the effect of gold nanoparticles on respiratory complex activities of isolated mitochondria demonstrated that the amino‐functionalized gold nanoparticles, but not the carboxy‐functionalized control particles, inhibit mitochondrial complex activities. We could further show that such direct inhibitory effect on mitochondrial complex activities also impairs mitochondrial respiration in intact THP‐1 cells. Inhibition of mitochondrial respiration further led to depolarization of the mitochondrial membrane, depletion of ATP, and cell death of THP‐1 cells and primary AML blasts.Hence, our findings confirm the high dependence of AML cells on oxidative phosphorylation. On that basis, our amino‐functionalized gold nanoparticles represent novel nanotherapeutics for a broad application in AML therapy, specifically targeting and inhibiting the sensitive energy production pathway in AML cells.Support or Funding InformationThis work was supported by the Volkswagen Foundation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Chracterization of THP-1 Cell Death Induced by Porphyromonas gingivalis Infection

Chracterization of THP-1 Cell Death Induced by Porphyromonas gingivalis Infection

Javamide-I-O-methyl ester increases p53 acetylation and induces cell death via activating caspase 3/7 in monocytic THP-1 cells

BackgroundJavamide-I and-II are phenolic amide compounds found in Coffea sp. Previous study suggested that javamide-II may be a potent compound with Sirt1/2 inhibition activity. PurposeHowever, the effects of javamide-I and the its O-methyl ester on Sirt inhibition, p53 acetylation and cell death have not been investigated. MethodsThe isolation and synthesis of javamide-I and its O-methyl ester analogue were confirmed by NMR. Their potential effects on Sirt1/2/3, p53 acetylation and cell death were examined using sirt assay, silico analysis, Western blot, caspase 3/7 and apoptotic assay methods. ResultsJavamide-I and its O-methyl ester demonstrated a similar inhibition pattern; Sirt1 (IC50 of 19µM) better than Sirt2 (IC50 of 104µM) and Sirt3 (IC50 of 160µM). However, javamide-I and its O-methyl ester were found to inhibit Sirt1 better than Sirt2, which is different from javamide-II able to inhibit Sirt2 stronger than Sirt1. In silico analysis, javamide-I and its O-methyl ester showed a competitive binding pattern against NAD+, which was also supported by the kinetic analysis with Ki=20.1µM for javamide-I and 19.5µM for the O-methyl ester. However, the O-methyl ester increased p53 acetylation better than javamide-I in monocytic THP-1 cells. Since caspase 3/7 activation is often followed by the p53 activation, we investigated their effects on caspase 3/7, and found that O-methyl ester again activated caspase 3/7 greater than javamide-I in the cells, eventually leading to apoptotic cell death. ConclusionThese data suggest that the O-methyl modification in javamide-I may play a critical role in increasing p53 acetylation, activating caspase, and eventually inducing the THP-1 cell death.

Effect of bisphosphonates on macrophagic THP-1 cell survival in bisphosphonate-related osteonecrosis of the jaw (BRONJ)

Immune deficiency and bacterial infection have been suggested to play a role in the pathophysiology of bisphosphonate-related osteonecrosis of the jaw (BRONJ). Zoledronate was previously found to promote THP-1 cell death. To examine this hypothesis with all commonly prescribed bisphosphonates, we tested the effect of (nitrogen-containing) ibandronate, risedronate, alendronate, pamidronate, and (non-nitrogen-containing) clodronate on macrophagic THP-1 cells. Activated THP-1 cells were exposed to .5 to 50 μM of nitrogen-containing bisphosphonates and .5 to 500 μM of clodronate. Cell adherence and survival were assessed in vitro using the xCELLigence real-time monitoring system. Results were confirmed histologically and verified with Live/Dead staining. All bisphosphonates inhibited THP-1 cell adherence and survival dose and time dependently, significant for zoledronate, alendronate, pamidronate, and clodronate in high concentrations (50 μM and 500 μM; P < .05). Low concentrations (0.5 μM) of risedronate, alendronate, and pamidronate prolonged the inflexion points of THP-1 cell survival compared with controls (P < .05). THP-1 cells exhibited no cytomorphologic changes at all concentrations. Commonly prescribed bisphosphonates inhibit the survival of macrophagic THP-1 cells dose-dependently without altering morphology. This may suggest a local immune dysfunction reflective of individual bisphosphonate potency leading to the pathogenesis of BRONJ.

Fumaric acid esters prevent the NLRP3 inflammasome-mediated and ATP-triggered pyroptosis of differentiated THP-1 cells

Fumaric acid esters (FAEs) exert therapeutic effects in patients with psoriasis and multiple sclerosis, however their mode of action remains elusive. Pyroptosis is a caspase-1-dependent pro-inflammatory form of programmed cell death, mediated by the activation of inflammasomes. To understand the pharmacological basis of the therapeutic effects of FAEs, the anti-pyroptotic activity of dimethyl fumarate (DMF) and its hydrolysis metabolite monomethyl fumarate (MMF) was studied in a model of NLRP3 inflammasome-mediated pyroptosis of human macrophages. Phorbol myristate acetate-differentiated THP-1 cells were exposed to lipopolysaccharide (5μg/ml; 4h), then pulsed with ATP (5mM; 1h). MMF, DMF, or parthenolide (positive control) were added 1h before the ATP pulse. The pyroptotic cell death was evaluated by morphological examination and quantified by measuring the lactate dehydrogenase leakage. The ATP-triggered death of THP-1 cells (60.4±4.0%) was significantly (P<0.01) prevented by DMF, in a time- and concentration-dependent manner (pIC50 and maximal effect were 6.6 and 67.6±1.2%, respectively). MMF was less efficacious than DMF. These effects were accompanied by a decreased intracellular activation of caspase-1 and interleukin-1β release from ATP-treated cells, thus suggesting that FAEs antagonise the effects of ATP by preventing the activation of the pyroptotic molecular cascade leading to cell death. These results indicate that FAEs are endowed with anti-pyroptotic activity, which may contribute to their therapeutic effects.

SomaticNLRP3mosaicism in Muckle-Wells syndrome. A genetic mechanism shared by different phenotypes of cryopyrin-associated periodic syndromes

: Familial cold autoinflammatory syndrome, Muckle-Wells syndrome (MWS), and chronic, infantile, neurological, cutaneous and articular (CINCA) syndrome are dominantly inherited autoinflammatory diseases associated to gain-of-function NLRP3 mutations and included in the cryopyrin-associated periodic syndromes (CAPS). A variable degree of somatic NLRP3 mosaicism has been detected in ≈35% of patients with CINCA. However, no data are currently available regarding the relevance of this mechanism in other CAPS phenotypes. To evaluate somatic NLRP3 mosaicism as the disease-causing mechanism in patients with clinical CAPS phenotypes other than CINCA and NLRP3 mutation-negative. NLRP3 analyses were performed by Sanger sequencing and by massively parallel sequencing. Apoptosis-associated Speck-like protein containing a CARD (ASC)-dependent nuclear factor kappa-light chain-enhancer of activated B cells (NF-κB) activation and transfection-induced THP-1 cell death assays determined the functional consequences of the detected variants. A variable degree (5.5-34.9%) of somatic NLRP3 mosaicism was detected in 12.5% of enrolled patients, all of them with a MWS phenotype. Six different missense variants, three novel (p.D303A, p.K355T and p.L411F), were identified. Bioinformatics and functional analyses confirmed that they were disease-causing, gain-of-function NLRP3 mutations. All patients treated with anti-interleukin1 drugs showed long-lasting positive responses. We herein show somatic NLRP3 mosaicism underlying MWS, probably representing a shared genetic mechanism in CAPS not restricted to CINCA syndrome. The data here described allowed definitive diagnoses of these patients, which had serious implications for gaining access to anti-interleukin 1 treatments under legal indication and for genetic counselling. The detection of somatic mosaicism is difficult when using conventional methods. Potential candidates should benefit from the use of modern genetic tools.

Differential effects of selenium (Se) on normal and malignant cells treated with chemotherapy (CT) and radiation (RT).

e13583 Background: Preclinical work has demonstrated that Se compounds potentiate anticancer effects of CT and RT while reducing normal tissue toxicities. The molecular basis for the therapeutic selectivity has yet to be fully elucidated but includes modulation of intracellular glutathione (GSH) concentrations, endoplasmic reticulum (ER) stress responses, DNA repair, induction of apoptosis and cellular resistance to CT and RT. Our aim was to evaluate the dose-response relationship of the Se compound methylseleninic acid (MSA) on molecular pathways involved in the response of normal and malignant cells to CT and RT. Methods: Peripheral blood mononuclear cells (PBMC) obtained from healthy blood donors and malignant THP-1 human monocytic leukaemia cells were exposed in vitro to MSA 2.5, 5 or 15 µM in varying combinations with MSA, RT, cisplatin (Pt), doxorubicin (Dox) and cytosine arabinoside (Ara-C). GSH concentration was measured by ELISA, DNA damage and repair by COMET assay, cell viability by MTT assay and ER stress response protein expression by western blotting. Results: MSA was selectively toxic to THP-1 cells and induced a protective increase in GSH in PBMC but a decrease in high concentrations within THP-1 cells. DNA damage induced by Ara-C or Dox in the COMET assay was significantly reduced by MSA in PBMC but increased in THP-1 cells. Cell death after 2 Gy RT was increased by all doses of MSA in THP-1 cells but only by the highest dose in PBMC. The cytotoxicity of Dox and Ara-C at sublethal doses was significantly enhanced by MSA in THP-1 cells and to a lesser extent in PBMC, but MSA increased cell death from Pt only in THP-1 cells. MSA induced a protective ER stress response in PBMC exposed to Ara-C but an apoptotic response in THP-1 cells. Conclusions: MSA at clinically-relevant concentrations had a differential effect on cell survival and death responses to RT and CT with relative protection of PBMC and enhanced death of THP-1 cells. Several mechanisms mediated this therapeutic selectivity and the dose-dependence of the Se effect varied between malignant and normal cells. These assays could potentially be used in clinical trials to evaluate pharmacodynamic markers of Se effects in conjunction with CT and/or RT.

A Significant Minority of Patients Prescribed Fracture Prevention Medication Lose Significant BMD on Repeat Testing

Immune deficiency and bacterial infection have been suggested to play a role in the pathophysiology of bisphosphonate-related osteonecrosis of the jaw (BRONJ). Zoledronate was previously found to promote THP-1 cell death. To examine this hypothesis with all commonly prescribed bisphosphonates, we tested the effect of (nitrogen-containing) ibandronate, risedronate, alendronate, pamidronate, and (non–nitrogen-containing) clodronate on macrophagic THP-1 cells.Activated THP-1 cells were exposed to .5 to 50 μM of nitrogen-containing bisphosphonates and .5 to 500 μM of clodronate. Cell adherence and survival were assessed in vitro using the xCELLigence real-time monitoring system. Results were confirmed histologically and verified with Live/Dead staining.All bisphosphonates inhibited THP-1 cell adherence and survival dose and time dependently, significant for zoledronate, alendronate, pamidronate, and clodronate in high concentrations (50 μM and 500 μM; P < .05). Low concentrations (0.5 μM) of risedronate, alendronate, and pamidronate prolonged the inflexion points of THP-1 cell survival compared with controls (P < .05). THP-1 cells exhibited no cytomorphologic changes at all concentrations.Commonly prescribed bisphosphonates inhibit the survival of macrophagic THP-1 cells dose-dependently without altering morphology. This may suggest a local immune dysfunction reflective of individual bisphosphonate potency leading to the pathogenesis of BRONJ.

Infection of human THP-1 cells with dormant Mycobacterium tuberculosis

Dormant, non-replicating Mycobacterium tuberculosis H37Rv strain cultured in hypoxic conditions was used to infect THP-1 cells. CFUs counting, Kinyoun staining and electron microscopy showed that dormant bacilli infected THP-1 cells at a rate similar to replicating M. tuberculosis, but failed to grow during the first 6 days of infection. The absence of growth was specific to the intracellular compartment, as demonstrated by efficient growth in liquid medium. Quantification of β-actin mRNA recovered from infected cells showed that, in contrast with log-phase bacteria, infection with dormant bacilli determined a reduced THP-1 cell death. Gene expression of intracellular non-replicating bacteria showed a pattern typical of a dormant state. Intracellular dormant bacteria induced the activation of genes associated to a proinflammatory response in THP-1 cells. Though, higher levels of TNFα, IL-1β and IL-8 mRNAs compared to aerobic H37Rv infected cells were not paralleled by increased cytokine accumulation in the supernatants. Moreover, dormant bacilli induced a higher expression of inducible cox-2 gene, accompanied by increased PGE2 secretion. Overall, our data describe a new model of in vitro infection using dormant M. tuberculosis that could provide the basis for understanding how non-replicating bacilli survive intracellularly and influence the maintenance of the hypoxic granuloma.

Inflammatory cytokine gene expression in THP‐1 cells exposed to Stachybotrys chartarum and Aspergillus versicolor

Very little is known about the mechanisms that occur in human cells upon exposure to fungi as well as their mycotoxins. A better understanding of toxin-regulated gene expression would be helpful to identify safe levels of exposure and could eventually be the basis for establishing guidelines for remediation scenarios following a water intrusion event. In this research, cytokine mRNA expression patterns were investigated in the human monocytic THP-1 cell line exposed to fungal extracts of various fragment sizes obtained from Stachybotrys chartarum RTI 5802 and/or Aspergillus versicolor RTI 3843, two common and well-studied mycotoxin producing fungi. Cytokine mRNA expression was generally upregulated 2-10 times following a 24 h exposure to fungal extracts. Expression of the proinflammatory interleukin-1β, interleukin-8, and tumor necrosis factor-α genes increased while the anti-inflammatory gene interleukin-10 also increased albeit at very low level, suggesting that negative feedback regulation mechanism of production of proinflammatory cytokines initiated upon 24 h of incubation. In addition, submicron size extracts of A. versicolor caused significant death of THP-1 cells, whereas extracts of S. chartarum caused no cell death while the mixture of the two fungi had an intermediate effect. There was no general correlation between gene expression and fragment sizes, which suggests that all submicron fragments may contribute to inflammatory response.

5068 Cardiac safety of non-pegylated liposomal doxorubicin in combination with docetaxel as 1st line treatment in locally advanced or metastatic HER2 negative breast cancer (MYOTAX study)

Immune deficiency and bacterial infection have been suggested to play a role in the pathophysiology of bisphosphonate-related osteonecrosis of the jaw (BRONJ). Zoledronate was previously found to promote THP-1 cell death. To examine this hypothesis with all commonly prescribed bisphosphonates, we tested the effect of (nitrogen-containing) ibandronate, risedronate, alendronate, pamidronate, and (non–nitrogen-containing) clodronate on macrophagic THP-1 cells.Activated THP-1 cells were exposed to .5 to 50 μM of nitrogen-containing bisphosphonates and .5 to 500 μM of clodronate. Cell adherence and survival were assessed in vitro using the xCELLigence real-time monitoring system. Results were confirmed histologically and verified with Live/Dead staining.All bisphosphonates inhibited THP-1 cell adherence and survival dose and time dependently, significant for zoledronate, alendronate, pamidronate, and clodronate in high concentrations (50 μM and 500 μM; P < .05). Low concentrations (0.5 μM) of risedronate, alendronate, and pamidronate prolonged the inflexion points of THP-1 cell survival compared with controls (P < .05). THP-1 cells exhibited no cytomorphologic changes at all concentrations.Commonly prescribed bisphosphonates inhibit the survival of macrophagic THP-1 cells dose-dependently without altering morphology. This may suggest a local immune dysfunction reflective of individual bisphosphonate potency leading to the pathogenesis of BRONJ.