Apoptotic Effects of a Thioether Analog of Vitamin K3 in a Human Leukemia Cell Line.

Research suggests that thioether analogs of vitamin K3 (VK3) can act to preserve the phosphorylation of epidermal growth factor receptors by blocking enzymes (phosphatases) responsible for their dephosphorylation. Additionally, these derivatives can induce apoptosis via mitogen-activated protein kinase and caspase-3 activation, inducing reactive oxygen species (ROS) production, and apoptosis. However, vitamin K1 exhibits only weak inhibition of phosphatase activity, while the ability of VK3 to cause oxidative DNA damage has raised concerns about carcinogenicity. Hence, in the current study, we designed, synthesized, and screened a number of VK3 analogs for their ability to enhance phosphorylation activity, without inducing off-target effects, such as DNA damage. 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay revealed that each analog produced a different level of cytotoxicity in the Jurkat human leukemia cell line; however, none elicited a cytotoxic effect that differed significantly from that of the control. Of the VK3 analogs, CPD5 exhibited the lowest EC50, and flow cytometry results showed that apoptosis was induced at final concentrations of ≥10 µM; hence, only 0.1, 1, and 10 µM were evaluated in subsequent assays. Furthermore, CPD5 did not cause vitamin K-attributed ROS generation and was found to be associated with a significant increase in caspase 3 expression, indicating that, of the synthesized thioether VK3 analogs, CPD5 was a more potent inducer of apoptosis than VK3. Hence, further elucidation of the apoptosis-inducing effect of CPD5 may reveal its efficacy in other neoplastic cells and its potential as a medication.

Reactive oxygen species-induced parthanatos of immunocytes by human cytomegalovirus-associated substance.

Previous studies have examined various immune evasion strategies of human cytomegalovirus (HCMV) to gain understanding of its pathogenesis. Although the mechanism that underlies immunocyte destruction near HCMV-infected lesions has yet to be established, it is here shown that substances produced by HCMV-infected cells induce death in several types of immunocytes, but not in fibroblasts or astrocytomas. These substances contain HCMV proteins and were termed HCMV-associated insoluble substance (HCMVAIS). The mechanism by which HCMVAIS induces cell death was characterized to improve understanding the death of immunocytes near HCMV-infected lesions. HCMVAIS were found to trigger production of intracellular nicotinamide adenine dinucleotide phosphate oxidase-derived reactive oxygen species (ROS), resulting in cell death, this effect being reversed following treatment with ROS inhibitors. Cell death was not induced in splenocytes from NOX-2 knockout mice. It was hypothesized that DNA damage induced by oxidative stress initiates poly ADP-ribose polymerase-1 (PARP-1)-mediated cell death, or parthanatos. HCMVAIS-induced cell death is accompanied by PARP-1 activation in a caspase-independent manner, nuclear translocation of apoptosis-inducing factor (AIF), and DNA fragmentation, which are typical features of parthanatos. Treatment with an AIF inhibitor decreased the rate of HCMVAIS-induced cell death, this being confirmed by hematoxylin and eosin staining; cell death in most HCMV-positive foci in serial section samples of a large intestine with HCMV infection was TUNEL-positive, cleaved caspase 3-negative and CD45-positive. Taken together, these data suggest that HCMV inhibits local immune responses via direct killing of immunocytes near HCMV-infected cells through ROS-induced parthanatos by HCMVAIS.

Titanium Ions Promote Exogenous Calcium-Dependent Calcium Influx in Activated Jurkat T Cells: A Possible Mechanism to Explain Its Immunostimulatory Properties.

Titanium and its alloys have been widely used in dental and orthopedic implants. Owing to the biotribocorrosion behavior of implants in simulated oral environment, Ti(IV) ions could be released into surrounding tissues. Current studies have found that Ti(IV) ions could affect the biological activities of immune cells in adjacent tissues and subsequently jeopardize the long-term performance of implant prostheses. However, the potential mechanism underlying its immunomodulatory properties remains unclear. Calcium signaling has been confirmed to be involved in regulation of lymphocyte immune function. Therefore, we hypothesize that Ti(IV) ions modulated T cell function through the change of intracellular calcium concentrations. This study is aimed at exploring the role of intracellular calcium responses in the modulatory effect of Ti(IV) ions on unactivated and phytohemagglutinin-activated Jurkat T cells. Here, we confirmed that Ti(IV) ions within a certain concentration range induced CD69 expression on both unactivated and activated T cells in our study. Additionally, the combined stimulation with Ti(IV) ions and PHA increased expression of IL-1ß, TNF-α, and RANKL. Furthermore, we found that treatment with Ti(IV) induced a transitory increase in the levels of [Ca2+]i in activated Jurkat cells, dependent on the presence of exogenous calcium. Treatment with different doses of Ti(IV) for 24 h significantly increased the levels of [Ca2+]i in the activated Jurkat cells in a dose-dependent manner, but had little effect in the unactivated cells. Treatment with Ti(IV) did not significantly affect the PLCγ1 activation and inositol-1,4,5-trisphosphate (IP3) secretion in Jurkat cells. Taken together, these data indicated that Ti(IV) enhanced calcium influx during the T cell activation, independent of IP3-mediated intracellular calcium release. Our work provides insights into the mechanism involved in the regulation of lymphocyte behaviors under the effect of Ti(IV) ions, which may help to develop therapeutic strategies for dental implant failures.

Bivalent 14-mer peptide ligands of CXCR4 with polyproline linkers with anti-chemotactic activity against Jurkat cells.

Interaction of CXCR4 with its endogenous ligand, stromal-cell derived factor-1 (SDF-1)/CXCL12, induces various physiological functions involving chemotaxis. Bivalent ligands with a polyproline helix bearing a cyclic pentapeptide, FC131, were previously shown to have higher binding affinities for CXCR4 than the corresponding monovalent ligands. Bivalent ligands based on a 14-mer peptide T140 derivative with polyproline linkers have been designed and synthesized. The activity of these peptides as well as the effect of bivalency of the ligand on CXCR4 binding has been assessed. The binding affinity of these series of bivalent ligands is increased as the linker length increases up to the 12-/15-mer proline linker. The inhibitory activity against chemotaxis on Jurkat cells also depends on the linker length. The T140-derived bivalent ligands with the 9- and 12-mer proline linkers showed the most effective inhibition against chemotaxis at 1000 nM, which is even higher than that of known CXCR4 antagonists in the monomer structure. The effective metastatic inhibition by bivalent T140 derivatives indicates the therapeutic potential. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

JNK/AP-1 activation contributes to tetrandrine resistance in T-cell acute lymphoblastic leukaemia.

T-cell acute lymphoblastic leukaemia (T-ALL) is a challenging malignancy with a high relapse rate attributed to drug resistance. Tetrandrine (TET), a bisbenzylisoquinoline alkaloid extracted from a Chinese herb, is a potential anti-cancer and anti-leukaemic drug. In this study we investigated the mechanisms of TET resistance in T-ALL cells in vitro. Among the four T-ALL cell lines tested, Jurkat and CEM cells exhibited the lowest and highest resistance to TET with IC50 values at 24 h of 4.31±0.12 and 16.53±3.32 µmol/L, respectively. When treated with TET, the activity of transcription factor activator protein 1 (AP-1) was significantly decreased in Jurkat cells but nearly constant in CEM cells. To avoid cell-specific variation in drug resistance and transcription factor activities, we established a TET-R Jurkat subclone with the estimated IC50 value of 10.90±.92 µmol/L by exposing the cells to increasing concentrations of TET. Interestingly, when treated with TET, TET-R Jurkat cells exhibited enhanced AP-1 and NF-κB activity, along with upregulation of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) signaling pathways, whereas the expression of P-gp was not altered. Selective inhibition of JNK but not ERK suppressed AP-1 activity and TET resistance in TET-R Jurkat cells and in CEM cells. These results demonstrate that Jurkat cells acquire TET resistance through activation of the JNK/AP-1 pathway but not through P-gp expression. The JNK/AP-1 pathway may be a potential therapeutic target in relapsed T-ALL.

Cloning and characterization of Halomonas elongata L-asparaginase, a promising chemotherapeutic agent.

L-asparaginase has been used in the treatment of patients with acute lymphoblastic leukemia (ALL) for more than 30 years. Rapid clearance of the enzyme from blood stream and its L-glutaminase-dependent neurotoxicity has led to searching for new L-asparaginases with more desirable properties. In the present study, L-asparaginase coding gene of Halomonas elongata was isolated, expressed in Escherichia coli, purified, and characterized. The purified protein was found to have a molecular mass of 39.5 kDa and 1000-folds more activity towards L-asparagine than L-glutamine. Enzyme-specific activity towards L-asparagine was determined to be 1510 U/mg, which is among the highest reported values for microbial L-asparaginases. K m , Vmax, and k cat values were 5.6 mM, 2.2 µmol/min, and 1.96 × 103 1/S, respectively. Optimum temperature was found to be 37 °C while the enzyme showed maximum activity at a wide pH range (from 6 to 9). Enzyme half-life in the presence of human serum at 37 °C was 90 min which is three times higher when compared with reported values for E. coli L-asparaginase. Enzyme showed cytotoxic effects against Jurkat and U937 cell lines with an IC50 of 2 and 1 U/ml, respectively. Also, no toxic effects on human erythrocytes and Chinese hamster ovary cell lines were detected, and just minor inhibitory effects on human umbilical vein endothelial cells were observed. This is the first report describing the therapeutic potentials of a recombinant L-asparaginase isolated from a halophilic bacterium as an anticancer agent.

Femtograms of Interferon-γ Suffice to Modulate the Behavior of Jurkat Cells: A New Light in Immunomodulation.

Since interferon-γ (IFN-γ) tunes both innate and adaptive immune systems, it was expected to enter clinical practice as an immunomodulatory drug. However, the use of IFN-γ has been limited by its dose-dependent side effects. Low-dose medicine, which is emerging as a novel strategy to treat diseases, might circumvent this restriction. Several clinical studies have proved the efficacy of therapies with a low dose of cytokines subjected to kinetic activation, while no in vitro data are available. To fill this gap, we investigated whether low concentrations, in the femtogram range, of kinetically activated IFN-γ modulate the behavior of Jurkat cells, a widely used experimental model that has importantly contributed to the present knowledge about T cell signaling. In parallel, IFN-γ in the nanogram range was used and shown to activate Signal transducer and activator of transcription (STAT)-1 and then to induce suppressor of cytokine signaling-1 (SOCS-1), which inhibits downstream signaling. When added together, femtograms of IFN-γ interfere with the transduction cascade activated by nanograms of IFN-γ by prolonging the activation of STAT-1 through the downregulation of SOCS-1. We conclude that femtograms of IFN-γ exert an immunomodulatory action in Jurkat cells.

A proteomic approach for the identification of immunotoxic properties of Tulipalin A.

The immune system is permanently exposed to several environmental influences that can have adverse effects on immune cells or organs leading to immunosuppression or inappropriate immunostimulation, called direct immunotoxicity. The natural compound Tulipalin A (TUPA), a lactone with α-methylene-γ-butyrolactone moiety, can influence the immune system and lead to allergic contact dermatitis. This in vitro study focused on effects of TUPA using two immune cell lines (Jurkat T cells and THP-1 monocytes). To evaluate the immunotoxic potential of the compound, a proteomic approach applying 2D gel electrophoresis and MALDI-TOF/TOF-MS in combination with metabolomic analysis was used after exposure of the cells to IC10 of TUPA. THP-1 cells showed a strong robustness to TUPA treatment since only five proteins were altered. In contrast, in Jurkat T cells an increase in the abundance of 66 proteins and a decrease of six proteins was determined. These intracellular proteins were mapped to biological processes. Especially an accumulation of chaperones and an influence on the purine synthesis were observed. The changes in purine synthesis were confirmed by metabolomic analysis. In conclusion, the data indicate possible target processes of low doses of TUPA in Jurkat T cells and provides knowledge of how TUPA affects the functionality of immune cells.

Vitamin E synthetic derivate-TPGS-selectively induces apoptosis in jurkat t cells via oxidative stress signaling pathways: implications for acute lymphoblastic leukemia.

D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) is a water-soluble derivative of natural vitamin E commonly used as a drug delivery agent. Recently, TPGS alone has been reported to induce cell death in lung, breast and prostate cancer. However, the effect of TPGS on cancer cell viability remains unclear. Thus, this study was aimed to evaluate the cytotoxic effect of TPGS on human periphral blood lymphocytes (PBL) and on T cell acute lymphocytic leukemia (ALL) Jurkat clone E6-1 cells and its possible mechanism of action. PBL and Jurkat cells were treated with TPGS (10, 20, 40, 60, and 80 µM), and morphological changes in the cell nucleus, mitochondrial membrane potential (ΔΨm), and intracellular reactive oxygen species levels were determined by immune-fluorescence microscopy and flow cytometry. Cellular apoptosis markers were also evaluated by immunocytochemistry. In this study, TPGS induced apoptotic cell death in Jurkat cells, but not in PBL, in a dose-response manner with increasing nuclear DNA fragmentation, increasing cell cycle arrest, and decreasing ΔΨm. Additionally, TPGS increased dichlorofluorescein fluorescence intensity, indicative of H2O2 production, in a dose-independent fashion. TPGS increased DJ-1 Cys(106)-sulfonate, as a marker of intracellular stress and induced the activation of NF-κB, p53 and c-Jun transcription factors. Additionally, it increased the expression of apoptotic markers Bcl-2 related pro-apoptotic proteins Bax and PUMAand activated caspase-3. The antioxidant N-acetyl-L-cysteine and known pharmacological inhibitors protected the cells from the TPGS induced effects. In conclusion, TPGS selectively induces apoptosis in Jurkat cells through two independent but complementary H2O2-mediated signaling pathways. Our findings support the use of TPGS as a potential treatment for ALL.

Baicalein suppresses the proliferation of acute T-lymphoblastic leukemia Jurkat cells by inhibiting the Wnt/ß-catenin signaling.

Although the response rates of chemotherapy in patients with acute T-lymphoblastic leukemia (T-ALL) have improved significantly, the outcome of these patients is still poor. Previous studies suggested that baicalein could inhibit the growth of several cancers, while its effect on T-ALL cells remains unclear. We used Jurkat cells as an in vitro model of T-ALL. Cell counting kit-8 assay and cytometric analysis with Annexin V-FITC/PI double staining were used to investigate the proliferation and apoptosis of Jurkat cells treated with increasing concentration of baicalein for indicated time. RT-PCR and western blotting was used to test the expression of Wnt/ß-catenin associated genes and proteins. In cell viability assay, baicalein could inhibit the proliferation of Jurkat cells both in dose- and time-dependent manners. In cell apoptosis assay, baicalein could stimulate apoptosis of Jurkat cells both in dose- and time-dependent manners. Moreover, we demonstrated that baicalein could down-regulated the mRNA and protein levels of ß-catenin and its widely accepted downstream targets (c-Myc, cyclin D1, and Axin2) in dose-dependent manners. These results proved that baicalein might be a potential choice for the treatment of T-ALL.

Reduction in Cell Viability and in Homeobox Protein Levels Following in Vitro Exposure to δ-tocopherol in Acute Myeloid Leukemia.

δ-Tocopherol (δ-T), the least prevalent tocopherol in our diet, was described to have a more potent anticancer activity in solid tumors compared to the other tocopherols. δ-T induces tumor cell death through peroxisome proliferator-activated receptor γ (PPAR-γ) induction, cyclin-D1 inhibition, and modulation of redox balance. Nevertheless, the role of δ-T in preventing or treating hematologic malignancies has not been studied. In this study, we screened the efficacy of δ-T against six cell lines representing a wide spectrum of hematologic malignancies: Jurkat (acute T-cell leukemia), K-562 (chronic myeloid leukemia), KG-1 [acute myeloid leukemia (AML)], THP-1 (acute monocytic leukemia), TOM-1 (acute lymphoblastic leukemia), and UMCL01-101 (AIDS-associated diffuse large B-cell lymphoma). Interestingly, the AML cell line KG-1 was the only one to be significantly affected at concentrations of δ-T as low as 20 µM. The antileukemic activity of δ-T in AML was verified in a set of primary cells collected from patients newly diagnosed with AML. Apoptotic induction and cell cycle arrest explained the efficacy of δ-T against KG-1 cells. The mechanism of cell growth inhibition of δ-T was through downregulation of cyclin-D1 and a set of homeobox proteins (HOXA9, PBX1, and Cdx2) that have a well-documented role in the pathobiology of AML.

Mapping Optimal Charge Density and Length of ROMP-Based PTDMs for siRNA Internalization.

A fundamental understanding of how polymer structure impacts internalization and delivery of biologically relevant cargoes, particularly small interfering ribonucleic acid (siRNA), is of critical importance to the successful design of improved delivery reagents. Herein we report the use of ring-opening metathesis polymerization (ROMP) methods to synthesize two series of guanidinium-rich protein transduction domain mimics (PTDMs): one based on an imide scaffold that contains one guanidinium moiety per repeat unit, and another based on a diester scaffold that contains two guanidinium moieties per repeat unit. By varying both the degree of polymerization and, in effect, the relative number of cationic charges in each PTDM, the performances of the two ROMP backbones for siRNA internalization were evaluated and compared. Internalization of fluorescently labeled siRNA into Jurkat T cells demonstrated that fluorescein isothiocyanate (FITC)-siRNA internalization had a charge content dependence, with PTDMs containing approximately 40 to 60 cationic charges facilitating the most internalization. Despite this charge content dependence, the imide scaffold yielded much lower viabilities in Jurkat T cells than the corresponding diester PTDMs with similar numbers of cationic charges, suggesting that the diester scaffold is preferred for siRNA internalization and delivery applications. These developments will not only improve our understanding of the structural factors necessary for optimal siRNA internalization, but will also guide the future development of optimized PTDMs for siRNA internalization and delivery.

Cellular determinants involving mitochondrial dysfunction, oxidative stress and apoptosis correlate with the synergic cytotoxicity of epigallocatechin-3-gallate and menadione in human leukemia Jurkat T cells.

We have investigated the growth-suppressive action of epigallocatechin-3-gallate (EGCG) on human leukemia Jurkat T cells. Results show a strong correlation between the dose-dependent reduction of clonogenic survival following acute EGCG treatments and the EGCG-induced decline of the mitochondrial level of Ca(2+). The cell killing ability of EGCG was synergistically enhanced by menadione. In addition, the cytotoxic effect of EGCG applied alone or in combination with menadione was accompanied by apoptosis induction. We also observed that in acute treatments EGCG displays strong antioxidant properties in the intracellular milieu, but concurrently triggers some oxidative stress generating mechanisms that can fully develop on a longer timescale. In parallel, EGCG dose-dependently induced mitochondrial depolarization during exposure, but this condition was subsequently reversed to a persistent hyperpolarized mitochondrial state that was dependent on the activity of respiratory Complex I. Fluorimetric measurements suggest that EGCG is a mitochondrial Complex III inhibitor and indicate that EGCG evokes a specific cellular fluorescence with emission at 400nm and two main excitation bands (at 330nm and 350nm) that may originate from a mitochondrial supercomplex containing dimeric Complex III and dimeric ATP-synthase, and therefore could provide a valuable means to characterize the functional properties of the respiratory chain.

Quambalarine B, a Secondary Metabolite from Quambalaria cyanescens with Potential Anticancer Properties.

Quambalarine B (QB) is a secondary metabolite produced by the basidiomycete Quambalaria cyanescens with potential anticancer activity. Here we report that QB at low micromolar concentration inhibits proliferation of several model leukemic cell lines (Jurkat, NALM6, and REH), whereas higher concentrations induce cell death. By contrast, the effect of QB on primary leukocytes (peripheral blood mononuclear cells) is significantly milder with lower toxicity and cytostatic activity. Moreover, QB inhibited expression of the C-MYC oncoprotein and mRNA expression of its target genes, LDHA, PKM2, and GLS. Finally, QB blocked the phosphorylation of P70S6K, a downstream effector kinase in mTOR signaling that regulates translation of C-MYC. This observation could explain the molecular mechanism behind the antiproliferative and cytotoxic effects of QB on leukemic cells. Altogether, our results establish QB as a promising molecule in anticancer treatment.

Cadmium-coordinated supramolecule suppresses tumor growth of T-cell leukemia in mice.

Cadmium is a toxic pollutant with occupational and environmental significance, due to its diverse toxic effects. Supramolecules that conjugate and decontaminate toxic metals have potential for use in treatment of cadmium intoxication. In addition, metal-coordinating ability has been postulated to contribute to the cytotoxic effects of anti-tumor agents such as cisplatin or bleomycin. Thiacalixarenes, cyclic oligomers of p-alkylphenol bridged by sulfur atoms, are supramolecules known to have potent coordinating ability to metal ions. In this study, we show that cadmium-coordinated thiacalix[4]arene tetrasulfate (TC4ATS-Cd) exhibits an anti-proliferative effect against T-cell leukemia cells. Cadmium exhibited cytotoxicity with IC50 values ranging from 36 to 129 µM against epithelia-derived cancer cell lines, while TC4ATS-Cd elicited no significant cytotoxicity (IC50 > 947 µM). However, a number of T-cell leukemia cell lines exhibited marked sensitivity to TC4ATS-Cd. In Jurkat cells, toxicity of TC4ATS-Cd occurred with an IC50 of 6.9 µM, which is comparable to that of 6.5 µM observed for cadmium alone. TC4ATS-Cd induced apoptotic cell death through activation of caspase-3 in Jurkat cells. In a xenograft model, TC4ATS-Cd (13 mg/kg) treatment significantly suppressed the tumor growth of Jurkat cells in mice. In addition, TC4ATS-Cd-treated mice exhibited significantly less cadmium accumulation in liver and kidney compared to equimolar cadmium-treated mice. These results suggest that cadmium-coordinated supramolecules may have therapeutic potential for treatment of T-cell leukemia.

Physiological levels of resveratrol metabolites are ineffective as anti-leukemia agents against Jurkat leukemia cells.

Dietary resveratrol is metabolically transformed in vivo by the intestine and liver to produce resveratrol glucuronides and sulfates in humans. Little is known about the anticancer activities of these metabolic products. The majority of in vitro studies have investigated effects of resveratrol aglycone at supraphysiological levels. Physiological levels of resveratrol-3-O-glucuronide, resveratrol-4'-O-glucuronide, and resveratrol-3-O-sulfate, the major in vivo metabolites of dietary resveratrol, were evaluated as anticancer agents against Jurkat T leukemia cells. Propidium iodide was use to measure cell death and changes in cell cycle, and the mitochondrial membrane dye JC-1 was used to measure changes in mitochondrial membrane potential by flow cytometry. PKH67 was used to evaluate changes in proliferation of the cells by flow cytometry. Jurkat cells were exposed to 0, 2.5, 5, 10, 15, and 20 µM of each resveratrol metabolite, which are concentrations achievable in vivo. None of the resveratrol metabolites were able to kill Jurkat T leukemia cells or alter cell cycle or proliferation at these concentrations. Only resveratrol-3-O-sulfate induced depolarization of mitochondrial membranes but without induction of cell death. These results suggest that the in vivo transformation of resveratrol to these glucuronide and sulfate metabolites renders these agents ineffective against T leukemia cells.

Cytotoxic activity of novel palladium-based compounds on leukemia cell lines.

Effective treatment methods for human leukemia are under development, but so far none of them have been found to be completely satisfactory. It was recently reported that palladium complexes have significant anticancer activity as well as lower toxicity compared with some clinically used chemotherapeutics. The anticancer activities of two novel palladium(II) complexes, [Pd(sac)(terpy)](sac)·4H2O and [PdCl(terpy)](sac)·2H2O, were tested against three human leukemia cell lines, Jurkat, MOLT-4, and THP-1, in comparison with cisplatin and adriamycin. The cytotoxic effect of the drugs was determined using the MTT assay. Cell death was assessed using fluorescein isothiocyanate-annexin/propidium iodide staining for flow cytometry. Furthermore, p53 phosphorylation, poly(ADP-ribose) polymerase cleavage, and Bax and Bcl-2 mRNA levels were examined to elucidate the mechanism of cell death induction. Both complexes exhibited a significant dose-dependent antigrowth effect in vitro. The complexes predominately induced apoptosis, but necrosis was also observed. In-vitro results have shown that palladium(II) complexes may be regarded as potential anticancer agents for treating human leukemia. Therefore, further analysis to determine the putative mechanism of action and in-vivo studies on animal models are warranted.

Development of novel DIF-1 derivatives that selectively suppress innate immune responses.

The multiple pharmacological activities of differentiation-inducing factor-1 (DIF-1) of the cellular slime mold Dictyostelium discoideum led us to examine the use of DIF-1 as a 'drug template' to develop promising seed compounds for drug discovery. DIF-1 and its derivatives were synthesized and evaluated for their regulatory activities in innate immune responses. We found two new derivatives (4d and 5e) with highly selective inhibitory activities against production of the antimicrobial peptide attacin in Drosophila S2 cells and against production of interleukin-2 in Jurkat cells.

Successful validation of genomic biomarkers for human immunotoxicity in Jurkat T cells in vitro.

Previously, we identified 25 classifier genes that were able to assess immunotoxicity using human Jurkat T cells. The present study aimed to validate these classifiers. For that purpose, Jurkat cells were exposed for 6 h to subcytotoxic doses of nine immunotoxicants, five non-immunotoxicants and four compounds for which human immunotoxicity has not yet been fully established. RNA was isolated and subjected to Fluidigm quantitative real time (qRT)-PCR analysis. The sensitivity, specificity and accuracy of the screening assay as based on the nine immunotoxicants and five non-immunotoxicants used in this study were 100%, 80% and 93%, respectively, which is better than the performance in our previous study. Only one compound was classified as false positive (benzo-e-pyrene). Of the four potential (non-)immunotoxicants, chlorantraniliprole and Hidrasec were classified immunotoxic and Sunset yellow and imidacloprid as non-immunotoxic. ToxPi analysis of the PCR data provided insight in the molecular pathways that were affected by the compounds. The immunotoxicants 2,3-dichloro-propanol and cypermethrin, although structurally different, affected protein metabolism and cholesterol biosynthesis and transport. In addition, four compounds, i.e. chlorpyrifos, aldicarb, benzo-e-pyrene and anti-CD3, affected genes in cholesterol metabolism and transport, protein metabolism and transcription regulation. qRT-PCR on eight additional genes coding for similar processes as defined in ToxPi analyzes, supported these results. In conclusion, the 25 immunotoxic classifiers performed very well in a screening with new non-immunotoxic and immunotoxic compounds. Therefore, the Jurkat screening assay has great promise to be applied within a tiered approach for animal free testing of human immunotoxicity.

Different storage conditions influence biocompatibility and physicochemical properties of iron oxide nanoparticles.

Superparamagnetic iron oxide nanoparticles (SPIONs) have attracted increasing attention in many biomedical fields. In magnetic drug targeting SPIONs are injected into a tumour supplying artery and accumulated inside the tumour with a magnet. The effectiveness of this therapy is thus dependent on magnetic properties, stability and biocompatibility of the particles. A good knowledge of the effect of storage conditions on those parameters is of utmost importance for the translation of the therapy concept into the clinic and for reproducibility in preclinical studies. Here, core shell SPIONs with a hybrid coating consisting of lauric acid and albumin were stored at different temperatures from 4 to 45 °C over twelve weeks and periodically tested for their physicochemical properties over time. Surprisingly, even at the highest storage temperature we did not observe denaturation of the protein or colloidal instability. However, the saturation magnetisation decreased by maximally 28.8% with clear correlation to time and storage temperature. Furthermore, the biocompatibility was clearly affected, as cellular uptake of the SPIONs into human T-lymphoma cells was crucially dependent on the storage conditions. Taken together, the results show that the particle properties undergo significant changes over time depending on the way they are stored.