Human Myelomonocytic Cell Line Research Articles

Abstract LB365: Generation of a novel severely immunodeficient Sprague Dawley rat model for xenograft studies

Abstract The recombinant-activating gene 2 (Rag2) plays a critical role in the recombination of the immunoglobulin and T cell receptor genes during the maturation of T and B cells. Loss of Rag2 protein leads to a lack of functional T and B cells, which eliminates the adaptive immune response. However, Rag2 deficiency alone may not guarantee the success of xenograft implantation, due to NK cells and other innate cells remaining. Il2rg is a key component of several cytokine receptors involved in differentiation and/or function of numerous adaptive and innate immune cells, including NK cells, mast cells, basophils, and dendritic cells. To generate a more severely immunodeficient rat model, both the Rag2 and Il2rg genes were knocked out in Sprague Dawley (SD) rats; this model was termed B-SDG rats. The immune profile and organ weights of thymus and spleen were investigated in B-SDG rats in comparison with Rag2-/- and wild-type SD rats. The spleen weights of both Rag2-/- and B-SDG rats were similar, but significantly reduced compared to wild-type SD rats. Notably, the thymus weights of B-SDG male rats were significantly smaller than those of Rag2-/- males. The immune cell profiles in thymus, spleen and blood were next evaluated by flow cytometry. A complete lack of B and T cells was observed in both Rag2-/- and B-SDG rats. B-SDG rats lacked CD161+ NK cells in the spleen and blood, which were maintained in Rag2-/- rats. Dendritic cells, monocytes, macrophages, and neutrophils were detected in all strains. In vivo xenograft implantation with human lung, bladder, breast, pancreas, acute monocytic and myelomonocytic leukemia tumor cell lines was next evaluated in B-SDG rats, which demonstrated successful engraftment. Furthermore, several efficacy studies were performed in B-SDG rats, including cisplatin chemotherapy on human breast invasive ductal carcinoma cell (HCC1954) and human large cell lung cancer cell (NCI-H460) xenografts, and a HER2-targeting ADC therapy was tested in B-SDG rats harboring human non-small cell lung cancer tumors (NCI-H1975). Taken together, these data confirm that B-SDG rats provide a powerful immunodeficient model for preclinical in vivo evaluation of novel drug candidates. Citation Format: Yanhui Nie, Meiqi Zhang, Wanling Zhong, Jia Feng, Bahetiyaer Huwatibieke, Zhaoxue Yu. Generation of a novel severely immunodeficient Sprague Dawley rat model for xenograft studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB365.

Design, optimization, production and activity testing of recombinant immunotoxins expressed in plants and plant cells for the treatment of monocytic leukemia

ABSTRACTAntibody–drug conjugates (ADCs) can improve therapeutic indices compared to plain monoclonal antibodies (mAbs). However, ADC synthesis is complex because the components are produced separately in CHO cells (mAb) and often by chemical synthesis (drug). They are individually purified, coupled, and then the ADC is purified, increasing production costs compared to regular mAbs. In contrast, it is easier to produce recombinant fusion proteins consisting of an antibody derivative, linker and proteinaceous toxin, i.e. a recombinant immunotoxin (RIT). Plants are capable of the post-translational modifications needed for functional antibodies and can also express active protein toxins such as the recombinant mistletoe lectin viscumin, which is not possible in prokaryotes and mammalian cells respectively. Here, we used Nicotiana benthamiana and N. tabacum plants as well as tobacco BY-2 cell-based plant cell packs (PCPs) to produce effective RITs targeting CD64 as required for the treatment of myelomonocytic leukemia. We compared RITs with different subcellular targeting signals, linkers, and proteinaceous toxins. The accumulation of selected candidates was improved to ~ 40 mg kg−1 wet biomass using a design of experiments approach, and corresponding proteins were isolated with a purity of ~ 80% using an optimized affinity chromatography method with an overall yield of ~ 84%. One anti-CD64 targeted viscumin-based drug candidate was characterized in terms of storage stability and cytotoxicity test in vitro using human myelomonocytic leukemia cell lines. We identified bottlenecks in the plant-based expression platform that require further improvement and assessed critical process parameters that should be considered during process development for plant-made RITs.

SYNTHESIS AND BIOLOGICAL ACTIVITY OF 2-(4-SUBSTITUTED BENZYLIDENE)- 7-METHYL-2H-THIAZOLO[3, 2-A] PYRIMIDINE-3,5-DIONES

The organosulphur thiazolo-pyrimidines are fused heterocyclic compounds that can be anticipated as 7-thio counterparts of the genuine purine bases such as guanine and adenine. They have attained a growing significance in the domain of drug chemistry because of their diverse pharmacological activities. In the current study, 2-substituted benzylidene-7-methyl-2H-thiazolo [3,2-a] pyrimidine-3,5-dione derivatives were synthesised. The synthetic compounds were tested against the human myelomonocytic leukaemia cell line (U-937) for their ability to inhibit cancer cell growth as well as against Gram negative E. coli (MTCC 40) and Gram positive S. aureus (MTCC 87) for their ability to inhibit bacterial growth. The amine and halogen containing compounds exhibited the strongest anticancer and antibacterial effects among all the derivatives in series (7a-j). Compounds 7h, 7e, 7a, 7b, 7c, 7i, and 7j displayed improved activity in both assays compared to standard andriyamycin and ciprofloxacin, whereas 7d, 7f, and 7g were shown to be moderately active. Through the use of IR, NMR and mass spectrum analyses, the molecular structures of the synthesized compounds were determined.

Expression of the RET Receptor Tyrosine Kinase and GDNFR-α in Normal and Leukemic Human Hematopoietic Cells and Stromal Cells of the Bone Marrow Microenvironment

AbstractThe RET proto-oncogene product is a receptor tyrosine kinase representing the signal-transducing molecule of a multisubunit surface receptor complex for the glial cell line-derived neurotrophic factor (GDNF ), in which a novel glycosyl-phosphatidylinositol (PI)-linked protein (termed GDNFR-α) acts as the ligand-binding component. We have analyzed expression of RET and GDNFR-α in purified normal hematolymphopoietic cells, leukemia/lymphoma cell lines, and 154 primary samples from patients with hematopoietic malignancies encompassing different lineages and differentiation stages. Relatively low amounts of RET mRNA were found in early CD34+ hematopoietic progenitors, but RET transcripts appeared to increase after myelomonocytic maturation. No expression of RET was found in peripheral blood and tissue B and T lymphocytes. Analysis of human myelomonocytic cell lines was overall consistent with results obtained on purified normal cells. Accordingly, RET expression was mainly confined to acute myeloid leukemias (AMLs) displaying either monocytic (French-American-British M4 and M5) or intermediate-mature myeloid (M2 and M3) phenotypes, being less frequently detected in early myeloid (M0 and M1) AMLs. In contrast, RET mRNA was sporadically detected in B-cell tumors, whereas, among T-cell malignancies, RET transcripts were mainly detected in cells of postthymic and mature T-cell phenotype. RET broad detection in primary tumors was not paralleled by the mutual expression of GDNFR-α, which was detected only in 2 isolated primary samples and in 3 leukemia/lymphoma cell lines. However, GDNFR-α transcripts, in the absence of RET mRNA, were found in normal bone marrow stromal cells (BMSC), in BM fibroblasts, and in two osteoblast cell lines previously described to support normal hematopoiesis. In the presence of GDNF-receptors derived from BMSC by PI-specific phospholipase C cleavage, GDNF efficiently bound RET-expressing AML blasts and was functionally active by reducing their clonogenic growth and triggering the monocytic maturation of leukemic cells.

The Role of Helicobacter pylori Proinflammatory Outer Membrane Protein and Propolis in Immunomodulation on U937 Macrophage Cell Model

Background: Regarding the important role of proinflammatory outer membrane protein (OipA) in the pathogenesis of Helicobacter pylori infection and immunomodulatory activity of propolis, we aimed to evaluate the immunogenicity effect of a purified recombinant OipA protein and propolis in the induction of two cytokines, interferon-gamma (IFN-γ) and interleukin-4 (IL-4), in a macrophage cell model. Materials and Methods: The recombinant protein used in the present study corresponding to the oipA expressing a 34-35 kDa protein. OipA protein was purified by Ni-NTA affinity chromatography. The purified OipA protein (2.5- 40 μg /mL) and the propolis ethanolic extract (5-40 μg/mL) were incubated with phorbol 12-myristate 13-acetate-treated human myelomonocytic cell line U937 cells. IL-4 and IFN-γ levels were measured after 48 hours of incubation using enzyme-linked immunosorbent assay. Results: The amounts of IL-4 and IFN-γ were significantly increased. The optimum concentration of OipA for the secretion of IL-4 was 5 μg/ml (P<0.0001). At higher concentrations, the amount of IL-4 diminished until suppression at 40 μg/mL. The optimum concentration of propolis, resulting in the most significant increased secretion of both IL-4 and IFN-γ was 40 μg/mL (P=0.0001 and P=0.0004). Conclusion: We found that an OipA concentration of 10 μg/mL was more effective for IFN-γ production; however, it was not effective for the high production of IL-4. Therefore, it is postulated that the OipA could mainly induce a Th1 response through the production of IFN-γ. We also observed propolis’s capability to induce IFN-γ production; however, the effective concentration for this was the same as for IL-4. Therefore, as an adjuvant, proper concentration of propolis is required for OipA to give the optimum response. [GMJ.2020;9:e1687]

The Role of Helicobacter pylori Proinflammatory Outer Membrane Protein and Propolis in Immunomodulation on U937 Macrophage Cell Model.

Background: Regarding the important role of proinflammatory outer membrane protein (OipA) in the pathogenesis of Helicobacter pylori infection and immunomodulatory activity of propolis, we aimed to evaluate the immunogenicity effect of a purified recombinant OipA protein and propolis in the induction of two cytokines, interferon-gamma (IFN-γ) and interleukin-4 (IL-4), in a macrophage cell model. Materials and Methods: The recombinant protein used in the present study corresponding to the oipA expressing a 34-35 kDa protein. OipA protein was purified by Ni-NTA affinity chromatography. The purified OipA protein (2.5- 40 μg /mL) and the propolis ethanolic extract (5-40 μg/mL) were incubated with phorbol 12-myristate 13-acetate-treated human myelomonocytic cell line U937 cells. IL-4 and IFN-γ levels were measured after 48 hours of incubation using enzyme-linked immunosorbent assay. Results: The amounts of IL-4 and IFN-γ were significantly increased. The optimum concentration of OipA for the secretion of IL-4 was 5 μg/ml (P<0.0001). At higher concentrations, the amount of IL-4 diminished until suppression at 40 μg/mL. The optimum concentration of propolis, resulting in the most significant increased secretion of both IL-4 and IFN-γ was 40 μg/mL (P=0.0001 and P=0.0004). Conclusion: We found that an OipA concentration of 10 μg/mL was more effective for IFN-γ production; however, it was not effective for the high production of IL-4. Therefore, it is postulated that the OipA could mainly induce a Th1 response through the production of IFN-γ. We also observed propolis’s capability to induce IFN-γ production; however, the effective concentration for this was the same as for IL-4. Therefore, as an adjuvant, proper concentration of propolis is required for OipA to give the optimum response.

The Role of Helicobacter Pylori Proinflammatory Outer Membrane Protein and Propolis in Immunomodulation on U937 Macrophage Cell Model

Background: Regarding the important role of proinflammatory outer membrane protein (OipA) in the pathogenesis of Helicobacter pylori infection and immunomodulatory activity of propolis, we aimed to evaluate the immunogenicity effect of a purified recombinant OipA protein and propolis in the induction of two cytokines, interferon-gamma (IFN-γ) and interleukin-4 (IL-4), in a macrophage cell model. Materials and Methods: The recombinant protein used in the present study corresponding to the oipA expressing a 34-35 kDa protein. OipA protein was purified by Ni-NTA affinity chromatography. The purified OipA protein (2.5- 40 μg /mL) and the propolis ethanolic extract (5-40 μg/mL) were incubated with phorbol 12-myristate 13-acetate-treated human myelomonocytic cell line U937 cells. IL-4 and IFN-γ levels were measured after 48 hours of incubation using enzyme-linked immunosorbent assay. Results: The amounts of IL-4 and IFN-γ were significantly increased. The optimum concentration of OipA for the secretion of IL-4 was 5 μg/ml (P&lt;0.0001). At higher concentrations, the amount of IL-4 diminished until suppression at 40 μg/mL. The optimum concentration of propolis, resulting in the most significant increased secretion of both IL-4 and IFN-γ was 40 μg/mL (P=0.0001 and P=0.0004). Conclusion: We found that an OipA concentration of 10 μg/mL was more effective for IFN-γ production; however, it was not effective for the high production of IL-4. Therefore, it is postulated that the OipA could mainly induce a Th1 response through the production of IFN-γ. We also observed propolis’s capability to induce IFN-γ production; however, the effective concentration for this was the same as for IL-4. Therefore, as an adjuvant, proper concentration of propolis is required for OipA to give the optimum response. [GMJ.2020;9:e1687]

Pharmacological Targets of Kaempferol Within Inflammatory Pathways-A Hint Towards the Central Role of Tryptophan Metabolism.

The flavonoid kaempferol is almost ubiquitously contained in edible and medicinal plants and exerts a broad range of interesting pharmacological activities. Interactions with central inflammatory processes can be exploited to treat or attenuate symptoms of disorders associated with chronic immune activation during infections, malignancies, and neurodegenerative or cardiovascular disorders. Many drugs, phytochemicals, and nutritional components target the catabolism of the essential amino acid tryptophan by indoleamine 2,3-dioxygenase 1 (IDO-1) for immunomodulation. We studied the effects of kaempferol by in vitro models with human peripheral blood mononuclear cells (PBMC) and THP-1 derived human myelomonocytic cell lines. Kaempferol suppressed interferon-γ dependent immunometabolic pathways: Formation of the oxidative stress biomarker neopterin and catabolism of tryptophan were inhibited dose-dependently in stimulated cells. In-silico docking studies revealed a potential interaction of kaempferol with the catalytic domain of IDO-1. Kaempferol stimulated nuclear factor kappa B (NF-κB) signaling in lipopolysaccharide (LPS)-treated THP-1 cells, thereby increasing the mRNA expression of interleukin (IL) 1 beta, tumor necrosis factor, and nuclear factor kappa B subunit 1, while IL6 was downregulated. Data suggest that concerted effects of kaempferol on multiple immunologically relevant targets are responsible for its immunomodulatory activity. However, the immunosuppressive effects may be more relevant in a T-cell dominated context.

CK2 Inhibitor CX4945 Shows Strong In Vivo Anti Leukemia Effect in AML Via Augmented Ikaros-Mediated Regulation of Global Epigenetic Landscape

Over-expression of Casein Kinase II (CK2) pro-oncogenic kinase in Acute Myelogenous Leukemia (AML) is associated with poor prognosis. Inhibition of CK2 by siRNAs or with the specific inhibitor, CX-4945, shows strong cytotoxic effects. CK2 is a ubiquitous, constitutively active serine/ threonine kinase which has been implicated in leukemia progression via multiple mechanisms. One of the well-established mechanism is CK2 mediated phosphorylation and impairment of tumor suppressor function of Ikaros transcription factor. Impaired function of Ikaros has been associated with the development of leukemia including AML. In B-cell lymphoblastic leukemia, CK2 inhibition has been shown to exert a therapeutic effect via restoration and/or enhancement of the tumor suppressor activity of the Ikaros protein. The mechanism of therapeutic action of CK2 inhibition in AML is largely unknown and in vivo efficacy CK2 inhibitors in AML has never been established. Here we report anti-leukemia effect of CX-4945 in preclinical models of AML and demonstrate mechanism of action of CK2 inhibitor via restoration of Ikaros driven global regulation of epigenetic landscape. In order to study the effect of CK2 inhibition on Ikaros function in AML, we used U937 - a human myelomonocytic leukemia cell line with high baseline CK2 expression. U937 cells were treated with specific CK2 inhibitor, CX4945 for 72 hours. CK2 activity and Ikaros phosphorylation was measured using CK2 kinase assay and in vivo labeling - radio immunoblot assay. Results showed significant decrease in CK2 activity and Ikaros phosphorylation with no change in overall expression of the Ikaros protein. Expression analysis using RNA sequencing showed that treatment with CX-4945 caused significant upregulation of genes controlling immunity, and inflammation; and downregulation of genes involved in nucleic acid metabolism, RNA processing, and translation. Analysis of global genome-wide Ikaros occupancy using Chromatin Immunoprecipitation followed by next generation sequencing (ChIP-seq) of CX4945 treated U937 cells demonstrated that CX-4945 treatment significantly increased the number of Ikaros binding sites as well as increased peak strength while minimally re-distributing Ikaros' global genomic occupancy. While increased binding to the Promoter, Gene Body, and Gene Desert elements in CX-4945-treated cells was similar, the increase in Ikaros' DNA binding to enhancers was particularly pronounced. Further analysis showed that enhanced Ikaros DNA binding following treatment with CX-4945, directly induces formation of de novo enhancers. In order to test whether enhanced DNA binding of Ikaros is accompanied by augmented Ikaros function in the global epigenetic regulation of gene expression. We determined and compared chromatin accessibility of U937 cells before and after treatment with CX-4945, using ATAC-seq. Results indicate that Ikaros-induced de novo open chromatin at distal regulatory regions controls genes involved in the negative regulation of biological processes and cellular metabolism. Overall, these data demonstrate that, augmented Ikaros DNA-binding following CK2 inhibition resulted in 1) Ikaros' pioneering activity, 2) Ikaros' ability to induce the de novo formation of enhancers and super-enhancers, and 3) Ikaros' ability to induce the de novo formation of active enhancers and to activate poised enhancers. Together, these data uncover novel Ikaros functions in regulating the epigenetic landscape and identifies CK2 as a critical regulator of Ikaros activity. Next, we treated AML xenograft model of luciferase labelled U937 cells with CX4945 via oral gavage at dose 200mg/kg/day for 3 weeks and demonstrated significantly lower leukemia burden in treated group as measured by decreased bio-luminescence imaging. In summary, these results demonstrate for the first time that CK2 inhibitor, CX-4945 has strong anti-leukemia effect in AML preclinical models. One of the mechanisms by which CX4945 exert a therapeutic effect in AML involves enhancing Ikaros' function as regulator of global epigenomic landscape. These results provide strong mechanistic basis to develop novel targeted combination therapies using CK2 inhibitors for treatment of AML. Further studies evaluating combination therapies using patient derived xenograft (PDX) models of AML are underway. Disclosures Payne: Elf Zone, Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

PB1982 AN IN VITRO STUDY OF THE INTERACTIONS BETWEEN THE CELL BASED CANCER VACCINE DCP‐001 AND ANTIGEN PRESENTING CELLS

Background:DCP‐001 is a whole cell‐based vaccine derived from the DCOne® human myelomonocytic leukemic cell line. DCOne cells endogenously express known and unknown tumour‐associated antigens. In the DCP‐001 manufacturing process, DCOne cells are differentiated and matured into cells with a mature dendritic cell phenotype. A phase I clinical trial demonstrated that the vaccine is safe and induces local and systemic cellular and humoral immune responses. DCP‐001 is currently studied in an international Phase II trial in AML patients ineligible for HSCT.Aims:To study the role of the interaction of whole cell‐based vaccine DCP‐001 with host immune cells.Methods:DCP‐001 and antigen presenting cell interaction studies were performed using flow cytometry and confocal microscopy. Cytokine/chemokine analyses were performed using luminex technology.Results:The strong immunogenicity of DCP‐001 is demonstrated by the broad range of chemokines and pro‐inflammatory cytokines released upon co‐culture of DCP‐001 with human peripheral blood mononuclear cells. Using different methods, we further demonstrate that antigen presenting cells efficiently process DCP‐001 through phagocytosis.Summary/Conclusion:These in vitro data suggest that in vivo, upon intradermal injection, DCP‐001 induces a strong inflammatory response, and is ingested by both resident as well as attracted host's APC, which subsequently prime tumour‐reactive T cells.

Optimized protocols for studying the NLRP3 inflammasome and assessment of potential targets of CP-453,773 in undifferentiated THP1 cells

The NLRP3 inflammasome is a complex multimeric signaling apparatus that regulates production of the pro-inflammatory cytokine IL-1β. To overcome both the variability among primary immune cells and the limitations of genetic manipulation of differentiated human or murine macrophages, we developed a simplified, reliable and relevant cell-based model for studying the NLRP3 inflammasome using the undifferentiated human myelomonocytic cell line THP1. Undifferentiated THP1 cells constitutively express NLRP3, and NLRP3 inflammasome activation occurred in response to canonical NLRP3 activation stimuli including nigericin, ATP, and urea crystals, culminating in pro-IL-1β cleavage, extracellular release of mature IL-1β, and pyroptosis. We used this THP1 cell system to investigate potential targets of the potent, NLRP3 inflammasome selective inhibitor CP-456,773. We optimized a viral shRNA transduction method for gene expression knockdown (KD), and the KD of NLRP3 itself eliminated inflammasome activation and IL-1β production. NLRP3 inflammasome activation and CP-453,773 pharmacology were not altered in ABCb7- or ABCb10-deficient THP1 cells, eliminating these gene products as candidate pharmacological targets of CP-453,773. For ABCb10, we confirmed our results using CRISPR/CAS9-mediated ABCb10 knockout (KO) THP1 sub-lines. In summary, undifferentiated THP1 cells are fully competent for activation of the NLRP3 inflammasome and production of IL-1β, without differentiation into macrophages, and we describe optimized KD and KO methodologies to manipulate gene expression in these cells. As an example of the utility of undifferentiated THP1 cells for investigations into the biology of the NLRP3 inflammasome, we have used this cell system to rule out ABCb7 and ABCb10 as potential targets of the NLRP3 inflammasome inhibitor CP-453,773.

The effect of alginate-gelatin encapsulation on the maturation of human myelomonocytic cell line U937

Today, many attempts have been collected in the field of tissue engineering for reconstitution of injured bone marrow capacity by transplantation of functional cell source. By having a three-dimensional condition, microcapsules are appropriate candidates for cells transplantation to target sites. Here, we examined the effect of alginate-gelatin microcapsules on functional maturation of human myelomonocytic cell line U937 after 7days in vitro. U937 cells were encapsulated by the mixture of alginate-gelatin and cultured for 7days. Trypan blue staining was used to show cell survival rate. Morphological changes were determined by haematoxylin and eosin staining. The expression of monocyte (CD14) and leukocyte (CD33) factors were measured by flow cytometry. The functional maturation of encapsulated cells was shown by immunocytochemistry targeting myeloperoxidase (MPO) activity and level of CD68. Transcription level of adhesion molecules CD68L, CD18, CD11b, and CD49d/VLA was detected by real-time polymerase chain reaction. In vivo constitutive capacity of encapsulated U937 was investigated in rabbits via administration to bone marrow. We showed enhanced U937 viability and monocyte and band cell-like appearance 7days after encapsulation. These changes coincided with increasing CD33 and CD14 levels and a decrease of CD15, confirming cell maturation (p<0.05). High level of MPO and CD68-positive cells showed the functional maturation of U937 cells into neutrophils and macrophages. Compared with that of nonencapsulated cells, the level of adhesion factor was up-regulated. We found labelled cells in the peripheral blood after cell transplantation to bone marrow. These data suggest that alginate-gelatin encapsulation of U937 cells promotes functional leukopoiesis and monocytopoiesis.

Rapid coupling between gravitational forces and the transcriptome in human myelomonocytic U937 cells

The gravitational force has been constant throughout Earth’s evolutionary history. Since the cell nucleus is subjected to permanent forces induced by Earth’s gravity, we addressed the question, if gene expression homeostasis is constantly shaped by the gravitational force on Earth. We therefore investigated the transcriptome in force-free conditions of microgravity, determined the time frame of initial gravitational force-transduction to the transcriptome and assessed the role of cation channels. We combined a parabolic flight experiment campaign with a suborbital ballistic rocket experiment employing the human myelomonocytic cell line U937 and analyzed the whole gene transcription by microarray, using rigorous controls for exclusion of effects not related to gravitational force and cross-validation through two fully independent research campaigns. Experiments with the wide range ion channel inhibitor SKF-96365 in combination with whole transcriptome analysis were conducted to study the functional role of ion channels in the transduction of gravitational forces at an integrative level. We detected profound alterations in the transcriptome already after 20 s of microgravity or hypergravity. In microgravity, 99.43% of all initially altered transcripts adapted after 5 min. In hypergravity, 98.93% of all initially altered transcripts adapted after 75 s. Only 2.4% of all microgravity-regulated transcripts were sensitive to the cation channel inhibitor SKF-96365. Inter-platform comparison of differentially regulated transcripts revealed 57 annotated gravity-sensitive transcripts. We assume that gravitational forces are rapidly and constantly transduced into the nucleus as omnipresent condition for nuclear and chromatin structure as well as homeostasis of gene expression.

Hydrogels in three-dimensional dendritic cell (MUTZ-3) culture as a scaffold to mimic human immuno competent subcutaneous tissue

The objective of this study was to develop a 3D cell culture model of the human subcutaneous tissue, allowing the prediction of the immunogenicity of subcutaneously injected therapeutic proteins. Several hydrogels were evaluated as scaffolds to mimic the human subcutaneous tissue in vitro. Cytocompatibility of the hydrogels with the human myelomonocytic cell line (MUTZ-3) was investigated, as well as their influence on cellular phenotype changes. Elastic Young’s moduli in compression of the hydrogels were measured by a texture analyser and compared to ex vivo human samples. MUTZ-3 cells were differentiated into dendritic cells before embedding in hydrogels. Agarose at various concentrations (0.5%, 0.35% and 0.25% w/v), Geltrex® matrix and HyStem™ scaffold (1% w/v) displayed a wide range of elastic Young’s moduli from 560 kPa to 49 kPa, compared to the reference value of 23 kPa obtained for human tissue. With the exception of HyStem™, good cytocompatibility of hydrogels was shown at the concentrations tested. An optimal combination of MUTZ-3 cells with 0.25% agarose or Geltrex® is suggested.

Baicalin Augments Hyperthermia-Induced Apoptosis in U937 Cells and Modulates the MAPK Pathway via ROS Generation

Background/Aims: Hyperthermia is a widely used therapeutic tool for cancer therapy and a well-known inducer of apoptosis. Although the flavonoid compound baicalin (BCN) is a potent anticancer agent for several human carcinomas, it is less potent in the human U937 myelomonocytic leukemia cell line. To explore any enhancing effects of BCN on hyperthermia-induced apoptosis, this study investigated the combined effects and apoptotic mechanisms of hyperthermia and BCN in U937 cells. Methods: U937 cells were heat treated at 44ºC for 12 min with or without pre-treatment with BCN (10-50 µM) and then incubated for 6 h at 37 ºC with 5% CO2 and 95% air. Cell viability was analyzed by Trypan blue exclusion assay. Apoptosis was examined by DNA fragmentation, fluorescence microscopy and flow cytometry. Generation of mitochondrial trans-membrane potential (MMP), mitochondrial calcium, and reactive oxygen species (ROS) was also detected by flow cytometry. The expression of proteins related to apoptosis and signaling pathways was determined by western blotting. Results: Hyperthermia alone did not reduce cell viability or induce notable levels of apoptosis, but combined hyperthermia and BCN treatment markedly augmented apoptosis by upregulating proapoptotic proteins and suppressing antiapoptotic proteins, culminating in caspase-3 activation. Mitochondrial transmembrane potential was significantly decreased, and generation of reactive oxygen species (ROS) and suppression of antioxidant enzymes were marked. Furthermore, with the combined treatment, the phosphorylated forms of JNK and p38 showed increased expression, whereas AKT was dephosphorylated. JNK-IN-8 (a JNK inhibitor) and NAC (a ROS scavenger) abrogated the apoptotic effects of the combined treatment, significantly protecting the cells and indicating the involvement of high ROS generation and the MAPK pathway in the underlying molecular mechanism. Conclusion: This study provides compelling evidence that hyperthermia, in combination with BCN, is a promising therapeutic strategy for enhancement of apoptosis and suggest a promising therapeutic approach for cancer.

Enhanced cell killing and apoptosis of oral squamous cell carcinoma cells with ultrasound in combination with cetuximab coated albumin microbubbles

Targeted microbubbles have the potential to be used for ultrasound (US) therapy and diagnosis of various cancers. In the present study, US was irradiated to oral squamous cell carcinoma cells (HSC-2) in the presence of cetuximab-coated albumin microbubbles (CCAM). Cell killing rate with US treatment at 0.9 W/cm2 and 1.0 W/cm2 in the presence of CCAM was greater compared to non-targeted albumin microbubbles (p < .05). On the other hand, selective cell killing was not observed in human myelomonocytic lymphoma cell line (U937) that had no affinity to cetuximab. Furthermore, US irradiation in the presence of CCAM showed a fivefold increase of cell apoptotic rate for HSC-2 cells (21.0 ± 3.8%) as compared to U937 cells (4.0 ± 0.8%). Time-signal intensity curve in a tissue phantom demonstrated clear visualisation of CCAM with conventional US imaging device. Our experiment verifies the hypothesis that CCAM was selective to HSC-2 cells and may be applied as a novel therapeutic/diagnostic microbubble for oral squamous cell carcinoma.

ChemInform Abstract: Aplysiasecosterol A: A 9,11‐Secosteroid with an Unprecedented Tricyclic γ‐Diketone Structure from the Sea Hare Aplysia kurodai.

AbstractThe title compound shows moderate cytotoxicity against human myelomonocytic leukemia cell line HL‐60.

Anti-proliferative activity of Monensin and its tertiary amide derivatives.

New tertiary amide derivatives of polyether ionophore Monensin A (MON) were synthesised and their anti-proliferative activity against cancer cell lines was studied. Very high activity (IC50=0.09 μM) and selectivity (SI=232) of MON against human biphenotypic myelomonocytic leukemia cell line (MV4-11) was demonstrated. The MON derivatives obtained exhibit interesting anti-proliferative activity, high selectivity index and also are able to break the drug-resistance of cancer cell line.

Pheophorbide a from Capsosiphon fulvescens Inhibits Advanced Glycation End Products Mediated Endothelial Dysfunction.

During hyperglycemia, the first step toward the formation of advanced glycation end products is the nonenzymatic glycation between the carbonyl group of a sugar and the primary amino group of a protein. Advanced glycation end products are then produced through more complex reactions. Reactive oxygen species derived from advanced glycation end products may play a key role in inflammation of the endothelium, leading to the complications seen in diabetes. Glycolaldehyde-induced advanced glycation end products have been reported to express proinflammatory cytokines, such as tumor necrosis factor-α and interleukin-1β. This study focused on Capsosiphon fulvescens, a Capsosiphonaceae type of green algae that has shown potential as a functional food material. Pheophorbide a, an anti-glycation compound, was isolated from C. fulvescens by extraction using a mixture of ethanol and water, followed by column fractionation of the resulting extract. The compound separated from C. fulvescens was identified by means of high-performance liquid chromatography combined with mass spectrometry. Pheophorbide a showed scavenging activity of the intracellular reactive oxygen species as well as monocyte adhesiveness inhibitory activity on the human myelomonocytic cell line (THP-1) and human umbilical vein endothelial cells cocultivation system. The mRNA levels of inflammation-related genes such as monocyte chemoattractant protein-1 and interleukin-6 were significantly decreased by pheophorbide a, and advanced glycation end products-stimulated tumor necrosis factor-α and interleukin-1β were downregulated as well. These results indicate that pheophorbide a has significant reactive oxygen species-scavenging activity, monocyte adhesive inhibitory activity, and downregulatory activity of cytokines related to inflammation affecting the endothelium. Pheophorbide a could therefore be a promising candidate for modulating endothelial cell dysfunction.

TGF-β decreases the stability of IL-18-induced IFN-γ mRNA through the expression of TGF-β-induced tristetraprolin in KG-1 cells.

We have previously reported that transforming growth factor-β (TGF-β) down-regulates interferon-γ (IFN-γ) production in an interleukin-18 (IL-18) treated mouse natural killer (NK) cell line, LNK5E6. In LNK5E6 cells, TGF-β exhibited no inhibition of the IL-18-induced transcription of IFN-γ, but did stimulate the degradation of IFN-γ mRNA induced by IL-18. In the present study, we investigated the mechanism of the down-regulatory effects of TGF-β on IFN-γ mRNA expression in a human myelomonocytic cell line, KG-1, which produces IFN-γ in response to IL-18 alone. Interestingly, IL-18 induced the production of the IFN-γ through the stabilization of IFN-γ mRNA, but not the enhanced transcription of IFN-γ gene. The stability of IFN-γ mRNA was regulated by mRNA destabilizing elements in the 3'untranslated region (UTR) of IFN-γ mRNA, especially adenylate-uridylate (AU)-rich elements (AREs) in the 5' half of 3'UTR. Tristetraprolin (TTP), one of the ARE-binding proteins, destabilizes IFN-γ mRNA, and IL-18 repressed the expression of TTP mRNA. Moreover, TGF-β repressed the IL-18-induced expression of IFN-γ mRNA through the induction of TTP mRNA to destabilize IFN-γ mRNA. Our data is the first to reveal that the crosstalk between IL-18 and TGF-β through the expression of TTP regulates the production of IFN-γ.