The Apoptotic Property of Nymphaea Caerulea Flower Extract on Acute Myeloid Leukaemia Cell Line, THP-1.

BACKGROUND: Acute Myeloid Leukaemia (AML) is considered to be an extremely heterogeneous malignancy of bone marrow and blood. The first line of therapy for AML is prolonged chemotherapy. Due to the presence of molecular heterogeneity in AML as confirmed by next-generation sequencing, researchers are planning to develop newer strategies of therapy. OBJECTIVE: In the present study we have explored the anti-cancer potentiality of the hydro-ethanolic extract (50% and 70%) of the whole flower of Nymphaea caerulea against the Acute Myeloid Leukaemia cell line, THP-1 with control of normal human kidney epithelial cell line (HEK 293). The present study is a novel contribution to the existing scientific knowledge as at present no study as an anti-leukaemic agent is available on N. caerulea (blue lotus) extract and exploring its action mechanism on in-vitro cell line model. METHODS: Some targeted cytokine and apoptotic genes genes to deduce the anti-cancer mechanism of action of the crude extract (hydro-ethanolic extract (50% and 70%) of the whole flower) were selected as Interferon (IFN) γ, Interleukins - IL-6, IL-8, IL- 10, IL-1ß, Transforming Growth Factor (TGF ß1), Tumor Necrosis Factor (TNF α), Caspase 3(CAS 3), Caspase 9 (CAS 9), CD95 (Fas), Tumor Necrosis Factor Receptor 1 (TNFRSF1A) to observe relative fold changes of the expression using Real-Time PCR with housekeeping gene ß-actin. Cellular cytopathic effect (CPE), cell viability assay by methylene blue assay, and cell cytotoxicity of the crude extract against the THP-1 cell line were also studied along with it's bio-active compositional analysis of the extract was explored using ultra-performance liquid chromatography followed by mass spectra. RESULTS: The N. caerulea flower extract is capable of inducing apoptosis in AML and it can balance cytokine alterations in such diseases. CONCLUSIONS: Nymphaea caerulea flower extract appears to be a good anti-leukemia agent.

Mitochondrial impairment related to the immunotoxicity of the herbicides clomazone, glyphosate and sulfentrazone in THP-1 cells.

Background: Pesticides are indispensable for the cultivation of crops, especially those of economic importance, such as soybeans. Data on the annual use of herbicides in crops show that they correspond to 50%, making it the most used in agriculture. Aim: Therefore, the aim of this study was to evaluate the toxicity of the three commercial herbicides (clomazone, glyphosate, and sulfentrazone) in THP-1 cells. Methods: Cells were incubated with 0-5,000 mg/L of the herbicides for 24 h at 37 °C for cytotoxicity evaluation. Additionally, a few toxicological pathways such as reactive species generation, mitochondrial impairment, and interleukin profile, which have been previously involved in the toxicity of pesticides, were also evaluated. Results: A potential immunotoxic effect of the herbicides on THP-1 cells was observed, especially glyphosate, as it is a powerful agent of cellular immunotoxicity. It was also possible to verify an increase in oxidative stress and IL-8 levels and mitochondrial dysfunction. Conclusion: All herbicides showed cytotoxic effects in THP-1 monocytes, which were related to mitochondrial impairment.

Characteristics of macrophage aggregates prepared by rotation culture and their response to polymeric materials.

Understanding the interaction between macrophages and biomaterials is important for the creation of new biomaterials and the development of technologies to control macrophage function. Since macrophages are strongly adhesive, caution is required when performing in vitro evaluations. Similarly, when THP-1 cells, macrophage precursor cells, are differentiated into macrophages using phorbol-12-myristate-13-acetate (PMA), it becomes difficult to detach them from the adherent substrate, which has been a problem on investigation of immunological responses to biomaterials. In this study, the interaction of THP-1 cell-differentiated macrophages with biomaterials was analyzed based on a new method of seeding THP-1 cells. THP-1 cells were cultured in static and rotation culture without and with PMA. In undifferentiated THP-1 cells, there was no change in cellular function between static and rotation cultures. In rotation culture with PMA, THP-1 cells differentiated and formed macrophage aggregates. IL-1ß and MRC1 expression in macrophage aggregates was examined after differentiation and M1/M2 polarization. Macrophage aggregates in rotation culture tended to be polarized toward M2 macrophages compared with those in static culture. In the evaluation of the responses of macrophage aggregates to several kinds of polymeric materials, macrophage aggregates showed different changes in MRC1 expression over time at 30, 50, and 70 rpm. Rotation speed of 30 rpm was considered most appropriate condition in that it gave stable results with the same trend as obtained with static culture. The use of macrophage aggregates obtained by rotational culture is expected to provide new insights into the evaluation of inflammatory properties of biomaterials.

Tp47-Induced Monocyte-Derived Microvesicles Promote the Adherence of THP-1 Cells to Human Umbilical Vein Endothelial Cells via an ERK1/2-NF-κB Signaling Cascade.

The Treponema pallidum membrane protein Tp47 induces immunocyte adherence to vascular cells and contributes to vascular inflammation. However, it is unclear whether microvesicles are functional inflammatory mediators between vascular cells and immunocytes. Microvesicles that were isolated from Tp47-treated THP-1 cells using differential centrifugation were subjected to adherence assays to determine the adhesion-promoting effect on human umbilical vein endothelial cells (HUVECs). Intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) levels in Tp47-induced microvesicle (Tp47-microvesicle)-treated HUVECs were measured, and the related intracellular signaling pathways of Tp47-microvesicle-induced monocyte adhesion were investigated. Tp47-microvesicles promoted THP-1 cell adhesion to HUVECs (P < 0.01) and upregulated ICAM-1 and VCAM-1 expression in HUVECs (P < 0.001). The adhesion of THP-1 cells to HUVECs was inhibited by anti-ICAM-1 and anti-VCAM-1 neutralizing antibodies. Tp47-microvesicle treatment of HUVECs activated the extracellular signal-regulated kinase 1/2 (ERK1/2) and NF-κB signaling pathways, whereas ERK1/2 and NF-κB inhibition suppressed the expression of ICAM-1 and VCAM-1 and significantly decreased the adhesion of THP-1 cells to HUVECs. IMPORTANCE Tp47-microvesicles promote the adhesion of THP-1 cells to HUVECs through the upregulation of ICAM-1 and VCAM-1 expression, which is mediated by the activation of the ERK1/2 and NF-κB pathways. These findings provide insight into the pathophysiology of syphilitic vascular inflammation.

Vitamin K3 Suppresses Pyroptosis in THP-1 Cells through Inhibition of NF-κB and JNK Signaling Pathways.

Vitamin K, a necessary nutritional supplement for human, has been found to exhibit anti-inflammatory activity. In the present study, we investigated the effects of vitamin K family on lipopolysaccharide (LPS) plus nigericin induced pyroptosis and explored the underlying mechanism of its action in THP-1 monocytes. Results showed that vitamin K3 treatment significantly suppressed THP-1 pyroptosis, but not vitamin K1 or K2, as evidenced by increased cell viability, reduced cellular lactate dehydrogenase (LDH) release and improved cell morphology. Vitamin K3 inhibited NLRP3 expression, caspase-1 activation, GSDMD cleavage and interleukin (IL)-1ß secretion in pyrophoric THP-1 cells. In addition, vitamin K3 inhibited the pro-inflammatory signaling pathways including nuclear factor-κB (NF-κB) and c-Jun N-terminal kinase (JNK). Vitamin K3 treatment also attenuated tissue damage and reduced serum LDH, IL-1ß and IL-6 levels in LPS-induced systemic inflammation of mice. The reduced myeloperoxidase (MPO) activityand F4/80 expression indicated that vitamin K3 effectively reduced the infiltration of neutrophils and macrophages. Moreover, NLRP3 expression in monocytes/macrophages were also decreased in vitamin K3-treatedmice after LPS challenge. These findings suggest that vitamin K3 potently alleviates systemic inflammation and organ injury via inhibition of pyroptosis in monocytes and may serve as a novel therapeutic strategy for patients with inflammatory diseases.

Immunopathological Inflammation in the Evolution of Mucositis and Peri-Implantitis.

The purpose of this study was to provide an immuno-mediated substantiation of the etiopathogenesis of mucositis and peri-implantitis based on the results of experimental, laboratory and clinical studies. The biopsy material was studied to identify impregnated nanoscale and microscale particles in the structure of pathological tissues by using X-ray microtomography and X-ray fluorescence analyses. Electron microscopy with energy-dispersive analysis identified the composition of supernatants containing nanoscale metal particles obtained from the surfaces of dental implants. The parameters of the nanoscale particles were determined by dynamic light scattering. Flow cytometry was used to study the effect of nanoscale particles on the ability to induce the activation and apoptosis of immunocompetent cells depending on the particles' concentrations during cultivation with the monocytic cell line THP-1 with the addition of inductors. An analysis of the laboratory results suggested the presence of dose-dependent activation, as well as early and late apoptosis of the immunocompetent cells. Activation and early and late apoptosis of a monocytic cell line when THP-1 was co-cultured with nanoscale metal particles in supernatants were shown for the first time. When human venous blood plasma was added, both activation and early and late apoptosis had a dose-dependent effect and differed from those of the control groups.

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OBJECTIVES: To explore the effect of polydatin on the proliferation and apoptosis of acute monocytic leukemia cell line THP-1 and the possible mechanism. METHODS: After THP-1 cells were treated with polydatin at gradient concentrations for 24 hours and 48 hours, their proliferation was determined by CCK-8 assay, and half maximal inhibitory concentration (IC50) was calculated. Logarithmically growing THP-1 cells were divided into two groups, a polydatin treatment group (treated with IC50 of polydatin) and a blank control group (treated without polydatin solution), and incubated for 48 hours. Cell apoptosis and cell cycle were measured by flow cytometry. The expression levels of PI3K, AKT, p-AKT, mTOR, p-mTOR, p70 S6K, and p-p70 S6K proteins were measured by Western blotting. RESULTS: After treatment with polydatin, the proliferation of THP-1 cells was strongly inhibited, and the IC50 at 48 hours was 1 800 µmol/L. After treatment with 1 800 µmol/L polydatin solution for 48 hours, the apoptosis rate of THP-1 cells increased significantly compared with the blank control group (P<0.05). The cell cycle was arrested in the G0/G1 and S phases, with a significantly increased proportion of cells in the G0/G1 phase and a significantly decreased proportion of cells in the S phase, as compared with the blank control group (P<0.05). The expression levels of PI3K, AKT, p-AKT, mTOR, p-mTOR, p70 S6K, and p-p70 S6K proteins decreased significantly compared with the blank control group (P<0.05). CONCLUSIONS: Polydatin can effectively inhibit the proliferation, block the cell cycle, and induce the apoptosis of THP-1 cells, which may be related to inhibition of the PI3K/AKT/mTOR signaling pathway.

A study of the THP-1 cell line as the potential biologics production platform with the emphasis on serum-free media substitution for economic expediency.

Cell cultures are frequently preferred in the industrial production of high value-added biopharmaceuticals on a large scale. In the production of biopharmaceuticals, the selection of the appropriate cell line, the cell culture medium, and the culture conditions are very important. In medical products to be offered for human use, authorized institutions do not allow the use of serum which increases the culture yield, added to the culture. For this reason, the cell lines to be used must be adapted to the serum-free medium. In this study, first, the direct and gradual adaptation of the THP-1 cell line, which has a high potential for use in biopharmaceutical production, to a locally produced chemically defined serum-free medium was carried out. Then, a comparison of the production efficiency in the shake flasks with the commercially available two different serum-free media was performed. Finally, for the first time, THP-1 cells were produced in spinner flasks and stirring tank bioreactor to simulate the large scale within the scope of this work.

Oncostatin M promotes the ox-LDL-induced activation of NLRP3 inflammasomes via the NF-κB pathway in THP-1 macrophages and promotes the progression of atherosclerosis.

Background: Oncostatin M (OSM) is reported to be involved in many stages of atherosclerosis, including endothelial dysfunction, chronic inflammation, and smooth muscle cell migration. This study explored the effects of OSM on foam cell formation and its corresponding molecular mechanisms. Methods: THP-1 cells were treated with phorbol-12-myristate-13-acetate (PMA) to induce macrophage differentiation and were then exposed to oxidized low-density lipoprotein (ox-LDL). OSM expression was analyzed by quantitative reverse transcription-polymerase chain reaction, Western blotting, and enzyme-linked immunosorbent assay (ELISA). OSM-specific small interfering RNAs (siRNAs) were transfected into THP-1 macrophages. The effects of OSM silencing were evaluated by Oil Red O staining, ELISA, and Western blotting. Moreover, the activation of NLR family pyrin domain containing 3 (NLRP3) inflammasomes was detected by western blotting and immunofluorescence. Results: OSM was highly expressed in THP-1 macrophages in a time- and dose-dependent fashion. Silencing OSM significantly reduced the total cholesterol content and Oil Red O staining levels in ox-LDL-treated macrophages. Silencing OSM significantly inhibited ox-LDL-induced cytokine release, including TNF-α, IL-1ß, IL-6, and IL-18. Ox-LDL activated p65 and NLRP3, which further induced caspase-1 cleavage, apoptosis-associated, speck-like protein containing a caspase-1 recruitment domain (ASC) upregulation, and gasdermin-D (GSDMD)-N fragmentation. Overexpression of NLRP3 significantly reversed the effects of OSM silencing on ox-LDL-induced foam cell formation and inflammation. Conclusions: OSM was highly expressed in the cell model of atherosclerosis. OSM has a promoting role in ox-LDL-induced foam cell formation and inflammation via the activation of p65-NLRP3 signaling pathways. Silencing OSM may be has benefit in treating atherosclerosis.

Comparative Analysis of Differentially Expressed Circular RNAs in Polarized Macrophages.

Macrophage polarization is a process that macrophages exert different functions according to surrounding micro-environment. Macrophages commonly exist in two distinct subsets: classically activated M1 macrophages and alternatively activated M2 macrophages. Circular RNAs (circRNAs) are a novel class of non-coding RNAs generated by back-splicing. Thousands of circRNAs were identified in different cells and tissues. Recent studies have revealed that circRNAs play a crucial role in regulating transcriptional and post-transcriptional gene expression. However, the effects and roles of circRNAs in macrophage polarization have not been well elucidated. Here, circRNAs expression profiles were determined in human THP-1 macrophages incubated in conditions causing activation toward M1 (interferon-γ + LPS) or M2 (interleukin-4) phenotypes. Overall, 9,720 circular RNA were detected from RNA sequencing data. Compared with M2 macrophages, a total of 140 circRNAs were aberrantly expressed in M1 macrophages, including 71 up-regulated circRNAs and 69 down-regulated circRNAs. Quantitative real-time PCR (qRT-PCR) results were generally consistent with the selected differentially expressed circRNAs. Gene Ontology (GO) and KEGG pathway analyses were used to predict biological functions and potential mechanisms of the host linear transcripts of these up-regulated and down-regulated circRNAs. Furthermore, we found that the expression level of circRNA-RNF19B (circRNF19B) in M1 macrophages was significantly higher than that in THP-1 macrophages and M2 macrophages. circRNF19B expression was increased when M2 converted to M1 whereas decreased when M1 converted to M2. Knockdown of circRNF19B following the activation of THP-1 cells using interferon-γ + LPS diminished the expression of M1 macrophages markers and elevated the expression of M2 macrophages markers. In conclusion, these data suggest the involvement of altered circRNAs expression patterns in macrophages exposure to different activating conditions. Circular RNAs may play important roles in regulating macrophage polarization.

Silencing YKL-40 gene can inhibit inflammatory factor expression and affects the effect of THP-1 cells on endometrial cancer.

PURPOSE: To investigate the effect of silencing the YKL-40 gene on the expression of inflammatory factors and the effect of silencing the YKL-40 gene of THP-1 cells on endometrial cancer. METHODS: We used a siRNA targeting a sequence in YKL-40 (si-YKL-40) to transfect HEC-1A and THP-1 cells. Quantitative real-time polymerase chain reaction assay was performed to investigate the mRNA levels of YKL-40, IL-8 and MMP-9 in HEC-1A and THP-1 cells. Migration, and invasion assays were performed to identify the effects of co-culture with THP-1 cells that silenced YKL-40 gene on the migration and invasion capacity of HEC-1A cells. Tube formation ability were detected by Matrigel-based angiogenesis assay. RESULTS: We successfully transfected HEC-1A and THP-1 cells with lentivirus to silence the YKL-40 gene. Compared with the blank control group and NC group, the expression of YKL-40, IL-8 and MMP-9 which were examined by qRT-PCR in YKL-40-siRNA group was significantly reduced in the two cell lines; after co-cultured with the supernatant of transfected THP-1 cells, the migration and invasion ability of HEC-1A cells in YKL-40-siRNA group was significantly reduced; the number of tubes in the YKL-40-siRNA group was significantly reduced, the spacing between the tubes was significantly increased, and the structure of tubes was incomplete. CONCLUSION: Silencing the YKL-40 gene in THP-1 cells can inhibit the expression of inflammatory factors, the invasion and migration of human endometrial cancer cells and the capacity of vitro angiogenic. And YKL-40 gene as a marker of inflammation may be an effective therapeutic target for endometrial cancer.

Activation of NLRP3 Inflammasome Complexes by Beta-Tricalcium Phosphate Particles and Stimulation of Immune Cell Migration in vivo.

Beta-tricalcium phosphate (ß-TCP) serves as a bone substitute in clinical practice because it is resorbable, biocompatible, osteointegrative, and osteoconductive. Particles of ß-TCP are also inflammatory mediators although the mechanism of this function has not been fully elucidated. Regardless, the ability of ß-TCP to stimulate the immune system might be useful for immunomodulation. The present study aimed to determine the effects of ß-TCP particles on NLR family pyrin domain containing 3 (NLRP3) inflammasome complexes. We found that ß-TCP activates NLRP3 inflammasomes, and increases interleukin (IL)-1ß production in primary cultured mouse dendritic cells (DCs) and macrophages, and human THP-1 cells in caspase-1 dependent manner. In THP-1 cells, ß-TCP increased also IL-18 production, and NLRP3 inflammasome activation by ß-TCP depended on phagocytosis, potassium efflux, and reactive oxygen species (ROS) generation. We also investigated the effects of ß-TCP in wild-type and NLRP3-deficient mice in vivo. Immune cell migration around subcutaneously injected ß-TCP particles was reduced in NLRP3-deficient mice. These findings suggest that the effects of ß-TCP particles in vivo are at least partly mediated by NLRP3 inflammasome complexes.

Effect of polydatin on the proliferation and apoptosis of THP-1 cells and the mechanism / 中国当代儿科杂志

OBJECTIVES@#To explore the effect of polydatin on the proliferation and apoptosis of acute monocytic leukemia cell line THP-1 and the possible mechanism.@*METHODS@#After THP-1 cells were treated with polydatin at gradient concentrations for 24 hours and 48 hours, their proliferation was determined by CCK-8 assay, and half maximal inhibitory concentration (IC50) was calculated. Logarithmically growing THP-1 cells were divided into two groups, a polydatin treatment group (treated with IC50 of polydatin) and a blank control group (treated without polydatin solution), and incubated for 48 hours. Cell apoptosis and cell cycle were measured by flow cytometry. The expression levels of PI3K, AKT, p-AKT, mTOR, p-mTOR, p70 S6K, and p-p70 S6K proteins were measured by Western blotting.@*RESULTS@#After treatment with polydatin, the proliferation of THP-1 cells was strongly inhibited, and the IC50 at 48 hours was 1 800 μmol/L. After treatment with 1 800 μmol/L polydatin solution for 48 hours, the apoptosis rate of THP-1 cells increased significantly compared with the blank control group (P<0.05). The cell cycle was arrested in the G0/G1 and S phases, with a significantly increased proportion of cells in the G0/G1 phase and a significantly decreased proportion of cells in the S phase, as compared with the blank control group (P<0.05). The expression levels of PI3K, AKT, p-AKT, mTOR, p-mTOR, p70 S6K, and p-p70 S6K proteins decreased significantly compared with the blank control group (P<0.05).@*CONCLUSIONS@#Polydatin can effectively inhibit the proliferation, block the cell cycle, and induce the apoptosis of THP-1 cells, which may be related to inhibition of the PI3K/AKT/mTOR signaling pathway.

Anti-Inflammatory Properties of Statin-Loaded Biodegradable Lecithin/Chitosan Nanoparticles: A Step Toward Nose-to-Brain Treatment of Neurodegenerative Diseases.

Nasal delivery has been indicated as one of the most interesting alternative routes for the brain delivery of neuroprotective drugs. Nanocarriers have emerged as a promising strategy for the delivery of neurotherapeutics across the nasal epithelia. In this work, hybrid lecithin/chitosan nanoparticles (LCNs) were proposed as a drug delivery platform for the nasal administration of simvastatin (SVT) for the treatment of neuroinflammatory diseases. The impact of SVT nanoencapsulation on its transport across the nasal epithelium was investigated, as well as the efficacy of SVT-LCNs in suppressing cytokines release in a cellular model of neuroinflammation. Drug release studies were performed in simulated nasal fluids to investigate SVT release from the nanoparticles under conditions mimicking the physiological environment present in the nasal cavity. It was observed that interaction of nanoparticles with a simulated nasal mucus decreased nanoparticle drug release and/or slowed drug diffusion. On the other hand, it was demonstrated that two antibacterial enzymes commonly present in the nasal secretions, lysozyme and phospholipase A2, promoted drug release from the nanocarrier. Indeed, an enzyme-triggered drug release was observed even in the presence of mucus, with a 5-fold increase in drug release from LCNs. Moreover, chitosan-coated nanoparticles enhanced SVT permeation across a human cell model of the nasal epithelium (×11). The nanoformulation pharmacological activity was assessed using an accepted model of microglia, obtained by activating the human macrophage cell line THP-1 with the Escherichia coli-derived lipopolysaccharide (LPS) as the pro-inflammatory stimulus. SVT-LCNs were demonstrated to suppress the pro-inflammatory signaling more efficiently than the simple drug solution (-75% for IL-6 and -27% for TNF-α vs. -47% and -15% at 10 µM concentration for SVT-LCNs and SVT solution, respectively). Moreover, neither cellular toxicity nor pro-inflammatory responses were evidenced for the treatment with the blank nanoparticles even after 36 h of incubation, indicating a good biocompatibility of the nanomedicine components in vitro. Due to their biocompatibility and ability to promote drug release and absorption at the biointerface, hybrid LCNs appear to be an ideal carrier for achieving nose-to-brain delivery of poorly water-soluble drugs such as SVT.

Tiaobu Feishen therapy inhibits inflammation induced by cigarette smoke extracts in a human monocyte/macrophage cell line.

OBJECTIVE: To study the mechanistic effects of Tiaobu Feishen therapy (TBFS) on inflammation induced by cigarette smoke extract (CSE) in a human monocyte/macrophage cell line. METHODS: The human monocyte/macrophage cell line THP-1 was stimulated with 10 % CSE in the presence or absence of Bufei Yishen formula (BYF), Bufei Jianpi formula (BJF) and Yiqi Zishen formula (YZF). All formulations contained serum. Pro-inflammatory cytokines were measured in the supernatants using enzyme-linked immunosorbent assay. The activity of STAT3 DNA binding was detected using electrophoretic mobility shift assay and janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway activation was assessed using Western blotting. RESULTS: The results showed that BYF, BJF and YZF treatment strongly decreased the CSE-induced secretion of interleukin (IL)-6, IL-8, tumor necrosis factor-α and matrix metalloproteinase-9 by THP-1 cells. Furthermore, BYF, BJF and YZF treatment attenuated STAT3 DNA binding capacity and JAK2 and STAT3 were shown to be phosphorylated. CONCLUSION: The data revealed that BYF, BJF and YZF effectively inhibited a CSE-induced inflammatory response in THP-1 cells by limiting activation of the JAK2/STAT3 pathway.

CRISPR-Cas9 Editing of Human Histone Deubiquitinase Gene USP16 in Human Monocytic Leukemia Cell Line THP-1.

USP16 is a histone deubiquitinase which facilitates G2/M transition during the cell cycle, regulates DNA damage repair and contributes to inducible gene expression. We mutated the USP16 gene in a high differentiation clone of the acute monocytic leukemia cell line THP-1 using the CRISPR-Cas9 system and generated four homozygous knockout clones. All were able to proliferate and to differentiate in response to phorbol ester (PMA) treatment. One line was highly proliferative prior to PMA treatment and shut down proliferation upon differentiation, like wild type. Three clones showed sustained expression of the progenitor cell marker MYB, indicating that differentiation had not completely blocked proliferation in these clones. Network analysis of transcriptomic differences among wild type, heterozygotes and homozygotes showed clusters of genes that were up- or down-regulated after differentiation in all cell lines. Prior to PMA treatment, the homozygous clones had lower levels than wild type of genes relating to metabolism and mitochondria, including SRPRB, encoding an interaction partner of USP16. There was also apparent loss of interferon signaling. In contrast, a number of genes were up-regulated in the homozygous cells compared to wild type at baseline, including other deubiquitinases (USP12, BAP1, and MYSM1). However, three homozygotes failed to fully induce USP3 during differentiation. Other network clusters showed effects prior to or after differentiation in the homozygous clones. Thus the removal of USP16 affected the transcriptome of the cells, although all these lines were able to survive, which suggests that the functions attributed to USP16 may be redundant. Our analysis indicates that the leukemic line can adapt to the extreme selection pressure applied by the loss of USP16, and the harsh conditions of the gene editing and selection protocol, through different compensatory pathways. Similar selection pressures occur during the evolution of a cancer in vivo, and our results can be seen as a case study in leukemic cell adaptation. USP16 has been considered a target for cancer chemotherapy, but our results suggest that treatment would select for escape mutants that are resistant to USP16 inhibitors.

IL-8 as a Potential Therapeutic Target for Periodontitis and Its Inhibition by Caffeic Acid Phenethyl Ester In Vitro.

Salivary levels of interleukin-8 (IL-8) are elevated in patients with periodontitis. Caffeic acid phenethyl ester (CAPE) improves the periodontal status in subjects. However, whether CAPE can reduce IL-8 expression is unclear. We collected saliva to determine proinflammatory cytokine levels and used subgingival calculus and surrounding tissues from patients with periodontitis for oral microbiota analysis via 16s ribosomal RNA gene sequencing. THP-1 cells were stimulated with sterile-filtered saliva from patients, and target gene/protein expression was assessed. IL-8 mRNA expression was analyzed in saliva-stimulated THP-1 cells treated with CAPE and the heme oxygenase-1 (HO-1) inhibitor tin-protoporphyrin (SnPP). In 72 symptomatic individuals, IL-8 was correlated with periodontal inflammation (bleeding on probing, r = 0.45; p < 0.001) and disease severity (bleeding on probing, r = 0.45; p < 0.001) but not with the four oral microbiota species tested. Reduced salivary IL-8 secretion was correlated with effective periodontitis treatment (r = 0.37, p = 0.0013). In THP-1 cells, saliva treatment induced high IL-8 expression and IKK2 and nuclear factor-κB (NF-κB) phosphorylation. However, the IKK inhibitor BMS-345541, NF-κB inhibitor BAY 11-7082, and CAPE attenuated saliva-induced IL-8 expression. CAPE induced HO-1 expression and inhibited IKK2, IκBα, and NF-κB phosphorylation. Blocking HO-1 decreased the anti-inflammatory activity of CAPE. The targeted suppression of IL-8 production using CAPE reduces inflammation and periodontitis.

Antiproliferative and cytotoxic activities of C-Geranylated flavonoids from Paulownia tomentosa Steud. Fruit.

Prenylated or geranylated flavonoids have been studied for their promising antiproliferative and cytotoxic activities. Twelve natural geranylated flavonoids (1-12) were isolated from the fruit of Paulownia tomentosa Steud. Their structures were elucidated using UV and IR spectroscopy, mass spectrometry, and 1D and 2D NMR spectroscopy. The absolute configurations were determined using NMR and circular dichroism. Seven of the compounds were characterized as new geranylated derivatives isolated from a natural source for the first time, namely 3'-O-methyl-5'-hydroxyisodiplacone (3), paulodiplacone A (5), tomentone II (6), tomentone B (7), tomentodiplacone P (8), paulodiplacone B (9), and tomentoflavone A (12). After 24 h of incubation at concentrations in the range 1-30 µM, the isolated compounds were tested for their antiproliferative and cytotoxic potentials against the human monocytic leukaemia cell line THP-1, using WST-1 and LDH assays, respectively. Almost all of the test compounds induced a concentration-dependent reduction in the metabolic activity of THP-1 cells and a concentration-dependent reduction in the cell viability. Diplacone (1) was the most potent antiproliferative and cytotoxic agent (IC50 9.31 ± 0.72 µM, LC50 18.01 ± 1.19 µM). 3'-O-Methyl-5'-hydroxydiplacone (2) showed relatively strong antiproliferative effect (IC50 12.61 ± 0.90 µM) and weaker cytotoxic activity (LC50 > 30 µM), indicating that it may serve as a potential lead compound for further testing. The structure-activity relationship for the 12 isolated compounds is discussed.

Isolation and Assessment of a Highly-Active Anti-Inflammatory Exopolysaccharide from Mycelial Fermentation of a Medicinal Fungus Cs-HK1.

The purpose of this work was to fractionate the complex exopolysaccharide (EPS) from a medicinal fungus Ophiocordyceps sinensis Cs-HK1 based on the molecular weight (MW) range and to assess the in vitro anti-inflammatory activity of different EPS fractions in THP-1 cell culture. The lower MW fraction (EPS-LM-1) showed a much higher anti-inflammatory activity. EPS-LM-1 was identified as a heteropolysaccharide consisting of mannose, glucose, and galactose residues with an average MW of 360 kDa. EPS-LM-1 significantly inhibited the lipopolysaccharide-induced inflammatory responses with the effective concentrations for 50% inhibition below 5 µg/mL on a few major proinflammatory markers. With such a notable in vitro anti-inflammatory activity, EPS-LM-1 is a promising candidate for the development of a new anti-inflammation therapy.