Acute Monocytic Leukemia Cell Research Articles

The P2X7 receptor mediates NLRP3-dependent IL-1β secretion and promotes phagocytosis in the macrophage response to Treponema pallidum

It is unclear whether P2X7 receptor (P2X7R) mediates NOD-like receptor family protein 3 (NLRP3)-dependent IL-1β secretion and spirochete phagocytosis in syphilis. This study was conducted to investigate the role of P2X7R in modifying NLRP3-dependent IL-1β secretion and regulating phagocytosis by Treponema pallidum (T. pallidum)-induced macrophages. Macrophages derived from a human acute monocytic leukemia cell line were cultured with T. pallidum. The activation of P2X7R in T. pallidum-treated macrophages occurred in a dose- and time-dependent manner. The P2X7R silencing group showed significantly decreased NLRP3 mRNA and protein levels (vs. the Tp group, P < 0.001). Similar results were observed for IL-1β secretion using ELISA (vs. the Tp group, P < 0.001). Furthermore, P2X7R siRNA transfection significantly decreased the percentage of spirochete-positive macrophages (29.73% vs. 70.83%, P < 0.001) and spirochete internalization (mean fluorescence intensity (MFI), 9.20 vs. 19.39, P < 0.001). This finding revealed that P2X7R played a role in the induction of NLRP3-dependent IL-1β secretion by T. pallidum-induced macrophages. Furthermore, we found that P2X7R plays an important role in IL-1β secretion and in the promotion of T. pallidum phagocytosis by macrophages. These results may not only contribute to our understanding of the immune mechanism that is active during T. pallidum infection but may also lay the groundwork for strategies to combat syphilis.

GP73 facilitates hepatitis B virus replication by repressing the NF-κB signaling pathway.

Hepatitis B virus (HBV) chronically infects approximately 350 million people worldwide, and 600 000 deaths are caused by HBV-related hepatic failure. Golgi protein 73 (GP73) is a serum biomarker for liver diseases, including chronic hepatitis B. Here, we determine the effect of HBV infection on GP73 production and characterized the role of GP73 in HBV replication. Initially, we show that GP73 is highly produced in the sera of HBV-positive patients with chronic liver diseases and in HBV-stimulated leukocytes. In addition, HBV stimulation promotes GP73 production in peripheral blood mononuclear cells isolated from healthy donors and in macrophages derived from human acute monocytic leukemia cells (THP-1). Notably, the hepatitis B surface antigen (HBsAg), but not HBV replication, is required for the activation of GP73 expression. Moreover, in HepG2 cells and Huh7 cells, GP73 facilitates HBV replication and represses nuclear factor kappa B p50 expression, which in turn represses HBV replication and GP73 expression. Finally, we demonstrate that GP73 facilitates HBV replication by repressing the innate immune response and the nuclear factor kappa B signaling pathway. Taken together, we revealed a distinct positive feedback mechanism between HBV replication and GP73 production and suggest that GP73 acts as a potential antiviral target for HBV infection.

Identification and functional study of immortalized mouse thymic epithelial cells

As the key cells in a three-dimensional scaffold within the thymus, Thymic epithelial cells (TECs) play critical roles in the homing, migration and differentiation of T cell precursors through adhesive interactions and the release of various cytokines. In this study, primary cultures of mouse TECs were isolated and identified with TEC-specific antibodies CK5 and CK8. These TECs were immortalized by retroviral transduction of simian virus (SV) 40 large T antigen. We then compared the functions of TECs and immortalized TECs (iTECs). Cell morphology and the proliferative capacity of TECs and iTECs were observed by inverted microscope photography and crystal violet assay after passage. A soft agar assay was then performed to observe their clone formation ability. The expression levels of epithelial cell related factors, such as IL-7, Lptin, Pax-9, Sema3A and et al., were detected by IF and qPCR. TECs were co-cultured with human acute monocytic leukemia cells (THP-1), and the effect of TECs on promoting THP-1 proliferation was observed with flow cytometry and CFSE labeling. Senescence-associated β-galactosidase assay was measured to detect the anti-aging capabilities of the cells. Cell cycle distribution was analyzed by propidium iodide (PI) staining, and paclitaxel (PTX)-induced apoptosis was detected by Annexin V-PI staining to evaluate the anti-apoptotic ability of the cells. Throughout, we found that the immortalized TECs still retain the characteristics of primary TECs, such as the morphology, function and epithelial characteristics; however, iTECs have stronger capabilities in proliferation and anti-aging. Our research suggests that the iTECs were successfully immortalized by SV40 large T antigen, and that the biological characteristics and functions of iTECs were similar to the original TECs. This immortalized cell can be used as an efficient cell model in functional research of the thymus substituting primary TECs with iTECs.

Cytotoxic and antiproliferative activities of amphibian (anuran) skin extracts on human acute monocytic leukemia cells

The use of preparations derived from frog skins for curative purposes antedates research history and is perpetuated in current medicine. The skins of anuran's (frogs and toads) are a rich source of compounds with a great importance in the search of antibiotics, analgesics, immunomodulators, enzymatic inhibitors and antitumoral agents applying to human health. Nowadays, cancer is the second most common cause of mortality with more than 8.2 million of deaths worldwide per year. Acute monocytic leukemia is the subtype M5 of acute myeloid leukemia (AML) a cancer type with reduced survival rates in patients. The monocyte to macrophage differentiation plays an essential role increasing the expansion of AML cell lines. Herein we studied the cytotoxic and antiproliferative activities of eleven amphibian species of three families belonging to Argentinean zones, against THP-1 monocytes and THP-1 macrophages acute monocytic leukemia cell lines. The evaluated species showed pronounced deleterious effects on acute monocytic leukemia THP-1 cell lines, reducing cell proliferation and inducing apoptosis, autophagy and in some cases cell aggregation. Being this work of great importance for the study of new natural anti-cancer compounds.

Effects of DNA Methylation on Leukemia Cell Proliferation.

In this study, we aimed to investigate the effect of DNA methyltransferase 1 gene (DNMT1) expression on leukemia cell proliferation. Following stimulation with interferon-α (IFN-α) or methylation inhibitor for three days, we evaluated changes in the DNMT1 expression levels, cell proliferation activity, and Bcl-2-Associated X Protein (BAX) expression levels in the chronic myelogenous leukemia cell line K562 and the acute monocytic leukemia cell line THP-1. DNMT1 expression levels and cell proliferation activity decreased in K562 and THP-1 cells, whereas BAX expression levels increased. These results suggest that the enzymatic activity of DNMT1 promotes the proliferation of tumor cells and that tumor cell proliferation can be suppressed by inhibiting DNMT1 enzymatic activity. Furthermore, because DNA methylation is associated with apoptosis, a process critical to cell growth and injury in leukemia, assessing DNMT1expression levels might help in treatment decisions for leukemia patients.

Transient Receptor Potential Ankyrin 1 Contributes to Lysophosphatidylcholine-Induced Intracellular Calcium Regulation and THP-1-Derived Macrophage Activation.

Lysophosphatidylcholine (LPC) is a major atherogenic lipid that stimulates an increase in mitochondrial reactive oxygen species (mtROS) and the release of cytokines under inflammasome activation. However, the potential receptors of LPC in macrophages are poorly understood. Members of the transient receptor potential (TRP) channel superfamily, which is crucially involved in transducing environmental irritant stimuli into nociceptor activity, are potential receptors of LPC. In this study, we investigated whether LPC can induce the activation of transient receptor potential ankyrin 1 (TRPA1), a member of the TRP superfamily. The functional expression of TRPA1 was first detected by quantitative real-time polymerase chain reaction (qRT-PCR), western blotting and calcium imaging in human acute monocytic leukemia cell line (THP-1)-derived macrophages. The mechanism by which LPC induces the activation of macrophages through TRPA1 was verified by cytoplasmic and mitochondrial calcium imaging, mtROS detection, a JC-1 assay, enzyme-linked immunosorbent assay, the CCK-8 assay and the lactate dehydrogenase (LDH) cytotoxic assay. LPC induced the activation of THP-1-derived macrophages via calcium influx, and this activation was suppressed by potent and selective inhibitors of TRPA1. These results indicated that TRPA1 can mediate mtROS generation, mitochondrial membrane depolarization, the secretion of IL-1β and cytotoxicity through cellular and mitochondrial Ca2+ influx in LPC-treated THP-1-derived macrophages. Therefore, the inhibition of TRPA1 may protect THP-1-derived macrophages against LPC-induced injury.

Inhibitory Effect of Eukaryotic Expression Vector Bearing TFPI-2 Gene on SHI-1 Cell Growth

To construct a eukaryotic expression vector of human tissue factor pathway inhibitor-2 (TFPI-2) and to investigate the effect of TFPI-2 gene on the growth of acute monocytic leukemia cell line (SHI-1). The cDNA of TFPI-2 was obtained by genetic chemical synthesis, the TFPI-2 gene and the linear vector fragment were ligated and inserted into the multiple cloning site of PEGFP-N1 vector, and the eukaryotic expression vector PEGFP-N1-TFPI-2 was transfected SHI-1 cells, then the obtained SHI-1 cells was observed by fluorescence microscopy; MTT assay was used to detect the effect of TFPI-2 gene on the relative growth rate of SHI-1 cells at the different time-point; RT-PCR was used to detect TFPI-2 mRNA expression levels in the cells of each group before and after TFPI-2 transfection; TFPI-2 protein expression was detected by Western blot. The cells which successfully transfected with PEGFP-N1-TFPI-2 vector were named as SHI-1-TFPI-2 (experimental group), and the cells transfected with the empty vector pEGFP-N1 and the untransfected cells were named as SHI-1-V and SHI-1-P and used as the control group. The human TFPI-2 gene eukaryotic expression vector PEGFP-N1-TFPI-2 was successfully constructed, then the transfected into SHI-1 cells, observed by fluorescence microscopy 24 hours later, as a result, the PEGFP-N1-TFPI-2 was successfully transferred into SHI-1 cells, and the number of fluorescent cells increased after 48 h and 72 h. RT-PCR showed that the gray scale ratio of TFPI-2 gene to β- actin in the experimental group was higher than that in the control group. The gray scale ratio was 0.51±0.04 in SHI-1-V group, 0.52±0.03 in SHI-1-P group, 0.87±0.08 in SHI-1-TFPI-2 group, and the difference between SHI-1-TFPI-2 and SHI-1-V, SHI-1-P group was statistically significant (P＜0.05). The expression of TFPI-2 gene in PEGFP-N1-TFPI-2 can inhibit the growth of SHI-1 cells, which provides a research direction for gene therapy of leukemia in the future.

Establishment and characterization of a DOT1L inhibitor-sensitive human acute monocytic leukemia cell line YBT-5 with a novel KMT2A-MLLT3 fusion.

Immortalized cell lines are useful for deciphering the pathogenesis of acute leukemia and developing novel therapeutic agents against this malignancy. In this study, a new human myeloid leukemia cell line YBT-5 was established. After more than 1-year cultivation from the bone marrow of a patient with acute monocytic leukemia, YBT cell line was established. Then a subclone, YBT-5, was isolated from YBT using single cell sorting. Morphological and cytogenetical characterizations of the YBT-5 cell line were determined by cytochemical staining, flow cytometry analysis, and karyotype analysis. Molecular features were identified by transcriptomic analysis and reverse transcription-polymerase chain reaction. To establish a tumor model, 5 × 106 YBT-5 cells were injected subcutaneously in nonobese diabetic/severe combined immune-deficiency (NOD/SCID) mice. DOT1L has been proposed as a potential therapeutic target for KMT2A-related leukemia; therefore, to explore the potential application of this new cell line, its sensitivity to a specific DOT1L inhibitor, EPZ004777 was measured ex vivo. The growth of YBT-5 does not depend on granulocyte-macrophage colony-stimulating factor. Cytochemical staining showed that α-naphthyl acetate esterase staining was positive and partially inhibited by sodium fluoride, while peroxidase staining was negative. Flow cytometry analysis of YBT-5 cells showed positive myeloid and monocytic markers. Karyotype analysis of YBT-5 showed 48,XY,+8,+8. The breakpoints between KMT2A exon 10 and exon 11 (KMT2A exon 10/11) and MLLT3 exon 5 and exon 6 (MLLT3 exon 5/6) were identified, which was different from all known breakpoint locations, and a novel fusion transcript KMT2A exon 10/MLLT3 exon 6 was formed. A tumor model was established successfully in NOD/SCID mice. EPZ004777 could inhibit the proliferation and induce the differentiation of YBT-5 cells. Therefore, a new acute monocytic leukemia cell line with clear biological and molecular features was established and may be used in the research and development of new agents targeting KMT2A-associated leukemia.

Antileukemic activity of novel adenosine derivatives

The present study investigated the effect of cladribine (CLA) and six of its derivatives containing a formamidine group at position 6 (CLA-FDM, CLA-FPAZ, CLA-FPIR, CLA-FPIP, CLA-FHEX, and CLA-FMOR) on acute promyelocytic, lymphoblastic, and acute monocytic leukemia cells. The role of ATR kinase in deoxycytidine kinase (dCK) activation in response to DNA damage was assessed. The presence of DNA lesions was assessed by measurement phosphorylation of H2AX and by using the alkaline comet assay with proteinase K post-treatment following assessment of the cell cycle. Apoptotic events such as alterations in intracellular calcium concentration, caspase-3/7 activity and increased sub-G1 cell population were measured. CLA derivatives were highly effective against leukemic cells, showing high cytotoxicity, causing DNA fragmentation, and inducing DNA-protein cross-links in leukemic cells. CLA-FMOR showed the highest efficacy. CLA derivatives increased the levels of intracellular calcium ions, caspase-3/7 and the percentage of sub-G1 apoptotic cells and blocked cells in the S phase of the cell cycle to a greater extent than free CLA. The selective ATR inhibitor VE-821 significantly suppressed the increase in dCK activity and decreased basal dCK activity. The present results suggested that ATR kinase controls dCK activity in response to synthetic CLA derivatives.

LPS induces inflammatory chemokines via TLR-4 signalling and enhances the Warburg Effect in THP-1 cells.

The Warburg Effect has emerged as a potential drug target because, in some cancer cell lines, it is sufficient to subvert it in order to kill cancer cells. It has also been shown that the Warburg Effect occurs in innate immune cells upon infection. Innate immune cells play critical roles in the tumour microenvironment but the Warburg Effect is not fully understood in monocytes. Furthermore, it is important to understand the impact of infections on key players in the tumour microenvironment because inflammatory conditions often precede carcinogenesis and mutated oncogenes induce inflammation. We investigated the metabolic programme in the acute monocytic leukaemia cell line, THP-1 in the presence and absence of lipopolysaccharide, mimicking bacterial infections. We found that stimulation of THP-1 cells by LPS induces a subset of pro-inflammatory chemokines and enhances the Warburg Effect. Surprisingly, perturbation of the Warburg Effect in these cells does not lead to cell death in contrast to what was observed in non-myeloid cancer cell lines in a previous study. These findings indicate that the Warburg Effect and inflammation are activated by bacterial lipopolysaccharide and may have a profound influence on the microenvironment.

MicroRNA‑20a negatively regulates the growth and osteoclastogenesis of THP‑1 cells by downregulating PPARγ.

The present study aimed to explore the mechanisms through which microRNA (miR)‑20a may be involved in the differentiation of THP‑1 human acute monocytic leukemia cells into osteoclasts. THP‑1 cells were differentiated into macrophages (osteoclast precursors) and subsequently into osteoclast cells. The expression levels of miR‑20a in THP‑1 cells were significantly reduced in a time‑dependent manner during phorbol‑12‑myristate‑13‑acetate (PMA), macrophage colony‑stimulating factor (M‑CSF) and receptor activator of nuclear factor‑κB ligand RANKL‑induced osteoclastogenesis. Following transfection with a miR‑20a mimics, the levels of miR‑20a in PMA‑treated THP‑1 cells increased more than 40‑fold as compared with expression in the control cells. In addition, the overexpression of miR‑20a inhibited proliferation, initiated S phase cell cycle arrest and induced apoptosis of PMA‑treated THP‑1 cells. Additionally, miR‑20a mimics treatment notably decreased the levels of tartrate‑resistant acid phosphatase, nuclear factor of activated T‑cells, cytoplasmic 1 and peroxisome proliferator‑activated receptor γ (PPARγ) during THP‑1 cell further differentiation progress. In summary, miR‑20a may negatively regulate the proliferation and osteoclastogenesis of THP‑1 cells during its osteoclast differentiation progress by downregulating PPARγ.

MicroRNA‑217 is involved in the progression of atherosclerosis through regulating inflammatory responses by targeting sirtuin 1.

Atherosclerosis is a chronic inflammatory disease, and it is a global clinical problem. The development of new and effective therapeutic targets for atherosclerosis is necessary. A number of microRNAs (miRNAs) have been demonstrated to serve a crucial role in atherosclerosis. However, the role of miRNA (miR)-217 in atherosclerosis remains unclear. Therefore, the aim of the present study was to investigate the role and mechanism of miR-217 in atherosclerosis. The level of miR-217 was detected in the blood of patients with atherosclerosis using reverse transcription-quantitative PCR. THP-1 acute monocytic leukemia cells were treated with oxidized low-density lipoprotein (ox-LDL) to develop an atherosclerotic cell model of macrophages. The relationship between miR-217 and sirtuin 1 (SIRT1) was determined by TargetScan and dual luciferase reporter assay. Cell apoptosis was measured by flow cytometry. Production of pro-inflammatory factors and triglyceride (TG) and total cholesterol (TC) levels were also determined. The results demonstrated that miR-217 was significantly upregulated in atherosclerosis. SIRT1 was demonstrated to be a direct target of miR-217 and was downregulated in atherosclerosis. Downregulation of miR-217 significantly inhibited ox-LDL-induced TG and TC level increase, cell apoptosis and the upregulation of the pro-inflammatory factors tumor necrosis factor α, interleukin (IL)-6 and IL-1β. Additionally, the SIRT1/AMP-activated protein kinase α/NF-κB pathway was at least partially involved in modulating the effects of miR-217 inhibition on THP-1 cells treated with ox-LDL. In addition, the effects of miR-217 downregulation on ox-LDL-treated THP-1 cells were eliminated by SIRT1 silencing. In conclusion, the results of the present study indicated that miR-217 downregulation may relieve atherosclerosis through the inhibition of macrophage apoptosis and inflammatory response by targeting SIRT1.

The regulation of inflammation-related genes after palmitic acid and DHA treatments is not mediated by DNA methylation.

Fatty acids (FAs) are known to participate in body inflammatory responses. In particular, saturated FAs such as palmitic acid (PA) induce inflammatory signals in macrophages, whereas polyunsaturated FAs, including docosahexaenoic acid (DHA), have been related to anti-inflammatory effects. Several studies have suggested a role of fatty acids on DNA methylation, epigenetically regulating gene expression in inflammation processes. Therefore, this study investigated the effect of PA and DHA on the inflammation-related genes on human macrophages. In addition, a second aim was to study the epigenetic mechanism underlying the effect of FAs on the inflammatory response. For these purposes, human acute monocytic leukaemia cells (THP-1) were differentiated into macrophages with 12-O-tetradecanoylphorbol-13-acetate (TPA), followed by an incubation with PA or DHA. At the end of the experiment, mRNA expression, protein secretion, and CpG methylation of the following inflammatory genes were analysed: interleukin 1 beta (IL1B), tumour necrosis factor (TNF), plasminogen activator inhibitor-1 (SERPINE1) and interleukin 18 (IL18). The results showed that the treatment with PA increased IL-18 and TNF-α production. Contrariwise, the supplementation with DHA reduced IL-18, TNF-α and PAI-1 secretion by macrophages. However, the incubation with these fatty acids did not apparently modify the DNA methylation status of the investigated genes in the screened CpG sites. This research reveals that PA induces important pro-inflammatory markers in human macrophages, whereas DHA decreases the inflammatory response. Apparently, DNA methylation is not directly involved in the fatty acid-mediated regulation of the expression of these inflammation-related genes.

Molecular hydrogen suppresses free-radical-induced cell death by mitigating fatty acid peroxidation and mitochondrial dysfunction.

Molecular hydrogen (H2) was believed to be an inert and nonfunctional molecule in mammalian cells; however, we overturned the concept by reporting the therapeutic effects of H2 against oxidative stress. Subsequently, extensive studies revealed multiple functions of H2 by exhibiting the efficacies of H2 in various animal models and clinical studies. Here, we investigated the effect of H2 on free-radical-induced cytotoxicity using tert-butyl hydroperoxide in a human acute monocytic leukemia cell line, THP-1. Cell membrane permeability was determined using lactate dehydrogenase release assay and Hoechst 33342 and propidium iodide staining. Fatty acid peroxidation and mitochondrial viability were measured using 2 kinds of fluorescent dyes, Liperfluo and C11-BODIPY, and using the alamarBlue assay based on the reduction of resazurin to resorufin by mainly mitochondrial succinate dehydrogenase, respectively. Mitochondrial membrane potential was evaluated using tetramethylrhodamine methyl ester. As a result, H2 protected the cultured cells against the cytotoxic effects induced by tert-butyl hydroperoxide; H2 suppressed cellular fatty acid peroxidation and cell membrane permeability, mitigated the decline in mitochondrial oxidoreductase activity and mitochondrial membrane potential, and protected cells against cell death evaluated using propidium iodide staining. These results suggested that H2 suppresses free-radical-induced cell death through protection against fatty acid peroxidation and mitochondrial dysfunction.

The N-acetylgalactosamine-transferase11 (GALNT11) activation in leukemia and the role of cyclophosphamide and cytarabine in expression of this enzyme

Background: We investigated the activation of N-acetylgalactosamine-transferase11 (GALNT11) and the role of cyclophosphamide and cytarabine in expression of this enzyme in AML and CML patients. Methods: GALNT11 activity and expression levels were evaluated in human chronic myelogenous leukemia cells (K562) and human acute monocytic leukemia cells (THP1) after incubation with different concentrations of cyclophosphamide (Cyclo) and cytarabine (Cyta). Anti-proliferative effects of Cyclo and Cyta were examined by MTT assay. Real time-PCR and ELISA assays were applied to investigate the expression and activity of GALNT11, respectively. Results: The IC50 values for Cyclo were 34.73±0.12 μM for K562 and 36.16±0.23 µM for THP1 cells. These valuses for Cyta were also 0.38±0.15 μM for K562 and 0.52±0.18 µM for THP1 cells . Conclusion: The activation and expression of GALNT11 increased in acute and chronic leukemia as glycosylation disorders. Also cyclophosphamide and specially cytarabine cause significant increase in activation and expression of GALNT11 which inhibits glycosylation and tumorogenesis dependent signaling pathway.

MiRNA143 Downregulated the Expression of Cyclophilin A to Inhibit the Progression of Atherosclerosis

To investigate the influence of microRNA (miR)-143 on atherosclerosis and its working mechanism via cyclophilin A. Human acute monocytic leukemia cells (human acute monocytic leukemia cells, human THP-1 monocytes) were treated with ox-LDL 80 mg/L for 48 h to establish a foam cell model. The modeling was evaluated by oil red O staining. The expressions levels of miRNA-143 and CyPA were detected by q-PCR. RNA extracted was confirmed by SiO2 nano magnetic bead. Dual-luciferase reporter gene assay was used to verify the targeted regulatory relationship between miRNA-143 and CyPA. Next, the cells were transfected with miR-143 mimics and treated with a CyPA inhibitor (cyclophilin A). The expression levels of CyPA and downstream matrix metalloproteinases-9 (MMP-9) in the downstream were detected by q-PCR and western blot, respectively. No or few lipid droplets were observed in the normal THP-1 cells. After treatment with ox-LDL for 48 h, a large amount of red-stained lipid was observed in the cytoplasm, indicating that the THP-1 derived foam cell models were successfully constructed. q-PCR results indicated that miRNA-143 was significantly downregulated in the THP-1-derived foam cell model, whereas CyPA was upregulated (P &lt; 0.05); the dual luciferase reporter gene assay was performed for miRNA-143 and 3'-UTR of CyPA, and the fluorescence intensity of the 3'-UTR vector group was significantly lower than that of the empty vector group (P &lt; 0.05). Fluorescence intensity detection of mutations at the target site showed that it had no effect was produced on the fluorescence intensity. This implied a negative regulatory effect conferred of miRNA-143 on CyPA; As compared to the model group, transfection with miRNA-143 mimics led to a significant downregulation of CyPA mRNA (P &lt; 0.05), which was accompanied by significant downregulation of both MMP-9 mRNA and protein (P &lt; 0.05). MMP-9 was also significantly downregulated upon the addition of the CyPA inhibitor cyclosporine A (P &lt; 0.05).

Abstract 4556: Targeting the TAM receptors on prostate cancer tumor-associated macrophages

Abstract Tumor growth and progression is influenced by the composition of the tumor microenvironment including host stromal cells and immune cells. In prostate cancer, a large proportion of the tumor volume is made up of macrophages, and high macrophage infiltrate correlates with poor prognosis. Macrophages can be polarized to various subtypes including M1 and M2 in response to stimuli in their environment. M1 macrophages have anti-tumor functions such as immune stimulation. M2 macrophages have pro-tumor functions such as promoting angiogenesis, extracellular matrix remodeling and immune suppression. The majority of prostate cancer tumor-associated macrophages exhibit M2-like characteristics. Thus, targeting M2 tumor-associated macrophages is a promising strategy for cancer therapy. M2 macrophages engage in efferocytosis, or phagocytosis of apoptotic cells. This process is an anti-inflammatory, immunosuppressive event that promotes tissue remodeling and tumor growth. The TAM receptors (Tyro3, Axl, MerTK) are a family of receptor tyrosine kinases whose role in efferocytosis has been well described in the literature. The TAM receptors bind phosphatidylserine on apoptotic cells using their ligands Gas6 and Protein S as bridging proteins. Following binding to phosphatidylserine, TAM receptor signaling induces a cytoskeleton rearrangement to engulf the apoptotic cell. Since efferocytosis is a tumor promoting process, we hypothesize targeting the TAM receptors on M2 macrophages may be a beneficial anti-cancer strategy. We are characterizing TAM receptor expression on M1 and M2 macrophages using two complementary in vitro methods: macrophage polarization of monocytes derived from healthy human donors and of the acute monocytic leukemia cell line THP-1. Cell surface markers of M1 macrophages (CD86) and M2 macrophages (CD163 and CD206) were measured to assess polarization by flow cytometry. We are currently further confirming the degree of polarization by measuring cytokine secretion and mRNA expression of M1- and M2-associated genes. We used flow cytometry analysis to assess TAM receptor expression on the in vitro polarized M1 and M2 macrophages. In the human monocyte derived model, we detected higher levels of expression of MerTK and Tyro3 on M2 macrophages in several biological replicates. We also found that M2 THP-1 macrophages express higher levels of MerTK and Tyro3 than M1 THP-1 macrophages, consistent with macrophages from the monocyte derived model. In conclusion, these data support the TAM receptors as a relevant therapeutic target for inhibiting M2 pro-tumor functions. Further work by qRT-PCR, western blot and immunofluorescence staining will be used to confirm higher levels of the MerTK and Tyro3 on M2 macrophages and further investigate expression of Axl on M1 and M2 macrophages. Citation Format: Kayla V. Myers, Kenneth J. Pienta. Targeting the TAM receptors on prostate cancer tumor-associated macrophages [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4556.

Curcumin can reverse the impairment of phagocytic function of macrophages induced by exosomes from patients with systemic lupus erythematosus

Objective To evaluate the effect of curcumin on phagocytic function of macrophages after the stimulation with exosomes from patients with systemic lupus erythematosus (SLE) . Methods From February 2016 to November 2017, 18 patients with active SLE and 18 healthy controls were enrolled from Guangzhou Institute of Dermatology and Sun Yat-sen Memorial Hospital, Sun Yat-sen University, and serum exosomes were extracted from these subjects. The exosomes from the healthy controls (CON-exo) and SLE patients (SLE-exo) as well as different concentrations (1, 5, 20 μmol/L) of curcumin were used alone or in combination to stimulate the acute monocytic leukemia cell line THP-1-derived macrophages. After coincubation of the stimulated macrophages with pHrodo™ Red indicator, flow cytometry was performed to determine the average fluorescence intensity, immunofluorescence staining to calculate the proportion of pHrodo™ Red-positive macrophages, and then the phagocytic activity of macrophages was assessed. Western blot analysis was conducted to determine the protein expression of CD14 in the stimulated macrophages. Statistical analysis was carried out by Student-t test for comparison between two groups, one-way analysis of variance for comparison among several groups, and least significant difference (LSD) -t test for multiple comparisons. Results Flow cytometry showed that there were significant differences in the relative fluorescence intensity of macrophages among the CON-exo group, CON-exo + 20 μmol/L curcumin group, SLE-exo group and SLE-exo + 20 μmol/L curcumin group (101.3% ± 14.05%, 94.27% ± 14.13%, 41.02% ± 9.54% and 87.33% ± 15.01%, respectively; F = 10.81, P < 0.01) , and immunofluorescence staining revealed that the proportion of pHrodo Red-positive macrophages significantly differed among the above 4 groups (82.16% ± 5.20%, 81.33% ± 4.51%, 54.20% ± 9.31% and 71.23% ± 5.43% respectively; F = 12.42, P < 0.01) . The fluorescence intensity of macrophages and proportion of pHrodo-positive macrophages were both significantly lower in the SLE-exo group than in the CON-exo group (t = 5.26, 5.35 respectively, both P < 0.01) and SLE-exo + 20 μmol/L curcumin group (t = 3.97, 3.26 respectively, both P < 0.05) . Western blot analysis showed that there were significant differences in the protein expression of CD14 among the CON-exo group, SLE-exo group, SLE-exo + 1 μmol/L curcumin group, SLE-exo + 5 μmol/L curcumin group and SLE-exo + 20 μmol/L curcumin group (96.33% ± 13.65%, 30.67% ± 5.86%, 45.24% ± 8.89%, 72.81% ± 6.62% and 90.67% ± 12.66% respectively; F = 24.57, P < 0.01) . The protein expression of CD14 was significantly lower in the SLE-exo group than in the CON-exo group (t = 8.06, P < 0.001) , SLE-exo + 5 μmol/L curcumin group and SLE-exo + 20 μmol/L curcumin group (t = 5.08, 7.38, both P < 0.001) , and the CD14 level showed an increasing trend along with the increase in the concentration of curcumin. Conclusion Exosomes from SLE patients markedly inhibit the phagocytosis of macrophages, while curcumin can reverse this inhibitory effect. Key words: Lupus erythematosus, systemic; Exosomes; Curcumin; Macrophages; Cytophagocytosis

Priming with GM-CSF instead of G-CSF enhances CAG-induced apoptosis of acute monocytic leukemia cells in vitro.

High expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor has been found in myelomonocytic or monocytic subtypes (M4/M5) of acute myeloid leukemia. Herein, we aimed to improve the effect of CAG [Ara-C, ACR, and G-CSF (granulocyte colony-stimulating factor)] regimen for acute monocytic leukemia by replacing G-CSF with GM-CSF. Results showed that the percentage of cells in S phase was higher with GM-CSF than with G-CSF treatment at 20ng/mL (P < 0.05). When THP-1 and SHI-1 cells were primed with 20ng/mL G-CSF or GM-CSF followed by Ara-C and ACR, cell proliferation rate in the CAGM (Ara-C, ACR, and GM-CSF) regimen was lower than in the CAG regimen (P < 0.05). Furthermore, CAGM regimen induced more obvious cell apoptosis than CAG regimen probably by reducing Bcl-2/Bax ratio (P < 0.05). Similar results were seen in primary cells from M5 patients. Collectively, our study suggests that priming with GM-CSF may be more effective than G-CSF in CAG regimen in acute monocytic leukemia.

Oxidized LDL, homocysteine, homocysteine thiolactone and advanced glycation end products act as pro-oxidant metabolites inducing cytokine release, macrophage infiltration and pro-angiogenic effect in ARPE-19 cells.

Age-related Macular Degeneration (AMD) is one of the major vision-threatening diseases of the eye. Oxidative stress is one of the key factors in the onset and progression of AMD. In this study, metabolites associated with AMD pathology more so at the systemic level namely, oxidized LDL (oxLDL), homocysteine (Hcy), homocysteine thiolactone (HCTL), advanced glycation end product (AGE) were evaluated for their pro-oxidant nature in a localized ocular environment based on in vitro studies in human retinal pigment epithelial cells (ARPE-19 cells). Human ARPE-19 cells were treated with pro-oxidants 50 μg/mL oxLDL, 500 μM Hcy, 500 nM HCTL, 100 μg/mL AGE, 200 μM H2O2 and 200 μM H2O2 with and without pre-treatment of 5 mM N-acetyl cysteine (NAC). The cytokines IL-6, IL-8 and vascular endothelial growth factor (VEGF) secreted from ARPE-19 cells exposed to pro-oxidants were estimated by ELISA. In vitro angiogenesis assay was performed with conditioned media of the pro-oxidant treated ARPE-19 cells in Geltrex-Matrigel coated 96-well plate. The human acute monocytic leukemia cell line (THP-1) was differentiated into macrophages and its migration in response to conditioned media of ARPE-19 cells insulted with the pro-oxidants was studied by transwell migration assay. Western blot was performed to detect the protein expression of Bax, Bcl-2 and NF-κB to assess apoptotic changes. The compounds involved in the study showed a significant increase in reactive oxygen species (ROS) generation in ARPE-19 cells (oxLDL; Hcy; AGE: p < 0.001 and HCTL: p < 0.05). NAC pre-treatment significantly lowered the oxidative stress brought about by pro-oxidants as seen by lowered ROS and MDA levels in the cells. Treatment with pro-oxidants significantly increased the secretion of IL-6 (oxLDL: p < 0.05; Hcy, HCTL and AGE: p < 0.01) and IL-8 cytokines (oxLDL: p < 0.05; HCTL: p <. 001 and AGE: p < 0.01) in ARPE-19 cells. Serum samples of AMD patients (n = 23) revealed significantly higher IL-6 and IL-8 levels compared to control subjects (n = 23) (IL6: p < 0.01 and IL8: p < 0.05). The pro-oxidants also promoted VEGF secretion by ARPE-19 cells compared to untreated control (oxLDL: p < 0.001; Hcy: p < 0.01; HCTL and AGE: p < 0.05). In vitro angiogenesis assay showed that the conditioned media significantly increased the tube formation in RF/6A endothelial cells. Transwell migration assay revealed significant infiltration of macrophages in response to pro-oxidants. We further demonstrated that the pro-oxidants increased the Bax/Bcl-2 ratio and increased the NF-κB activation resulting in pro-apoptotic changes in ARPE-19 cells. Thus, oxLDL, Hcy, HCTL and AGE act as pro-oxidant metabolites in RPE that promote AMD through oxidative stress, inflammation, chemotaxis and neovascularization.