THP-1 Tumor Cells Research Articles

Design, synthesis and biological evaluation of novel benzofuran derivatives as potent LSD1 inhibitors.

Lysine-specific demethylase 1 (LSD1) is a FAD-dependent enzyme, which has been proposed as a promising target for therapeutic cancer. Herein, a series of benzofuran derivatives were designed, synthesized and biochemical evaluated as novel LSD1 inhibitors based on scaffold hopping and conformational restriction strategy. Most of the compounds potently suppressed the enzymatic activities of LSD1 and potently inhibited tumor cells proliferation. In particular, the representative compound 17i exhibited excellent LSD1 inhibition at the molecular levels with IC50=0.065μM, as well as anti-proliferation against MCF-7, MGC-803, H460, A549 and THP-1 tumor cells with IC50 values of 2.90±0.32, 5.85±0.35, 2.06±0.27, 5.74±1.03 and 6.15±0.49μM, respectively. The binding modes of these compounds were rationalized by molecular docking. Meanwhile, a preliminary druggability evaluation showed that compound 17i displayed favorable liver microsomal stability and weak inhibitory activity against CYPs at 10μM. Remarkably, H460 xenograft tumors studies revealed that 17i demonstrated robust invivo antitumor efficacy without significant side effects. All the results demonstrated that compound 17i could represent a promising lead for further development.

Differential Localization and Invasion of Tumor Cells in Mouse Models of Human and Murine Leukemias.

Leukemias are refractory hematopoietic malignancies, for which the development of new therapeutic agents requires in vivo studies using tumor-bearing mouse models. Although several organs are commonly examined in such studies to evaluate the disease course, the effectiveness of interventions and the localization of tumor cells in the affected organs are still unclear. In this study, we histologically examined the distribution of leukemia cells in several organs using two leukemic mouse models produced by the administration of two cell lines (THP-1, a human myelomonocytic leukemia, and A20, a mouse B cell leukemia/lymphoma) to severe immunodeficient mice. Survival of the mice depended on the tumor burden. Although A20 and THP-1 tumor cells massively infiltrated the parenchyma of the liver and spleen at 21 days after transplantation, A20 cells were hardly found in connective tissues in Glisson’s capsule in the liver as compared with THP-1 cells. In the bone marrow, there was more severe infiltration of A20 cells than THP-1 cells. THP-1 and A20 cells were widely spread in the lungs, but were rarely observed in the small intestine. These findings suggest that each leukemia model has a unique localization of tumor cells in several affected organs, which could critically affect the disease course and the efficacy of therapeutic agents, including cellular immunotherapies.

Abstract B068: An in vitro cellular functional STING pathway assay to measure the potency of STING agonists, using interferon-β response

Abstract Introduction and Relevance: We have developed an in vitro cellular functional STING pathway assay that can be used to screen for potential therapeutic agents in drug discovery. Stimulator of interferon genes (STING, or TMEM173) is an endoplasmic reticulum-associated protein and cytosolic DNA sensor, which plays a critical role in the innate and adaptive arm of the immune system. It is highly expressed in antigen-presenting cells (APC’s), such as macrophages and dendritic cells, as well as plasmacytoid dendritic cells, myeloid-derived suppressor cells, T-cells, and various endothelial or epithelial subtypes. The STING pathway is spontaneously activated by cyclic dinucleotides, a product derived from the intracellular enzyme, cyclic GMP-AMP synthase (cGAS), upon invasion by pathogens and exposure to self-DNA, which leads to the production of type I interferons and proinflammatory cytokines. Recent studies in the tumor microenvironment have suggested that STING pathway activation in APC’s leads to the production of interferon-β (IFNβ) and spontaneous generation of a strong antitumor CD8+ T-cell response. Preclinical models with STING agonists have shown significant regression of established tumors, which has subsequently resulted in phase I clinical trials of these agents. Based on the collective evidence, pharmacologic STING agonists are now being developed as a potential new approach for cancer immunotherapy. Within this context, we investigated whether STING pathway activation in peripheral blood mononuclear cells (PBMCs) and the monocytic tumor cell line, THP-1, could be used to develop an assay to assist the discovery of novel STING agonist therapeutics. Experimental Procedures: Freshly isolated human PBMCs, or THP-1 cells, are plated and treated with STING agonists (e.g., 2’3’-cGAMP), or test agents, over a range of concentrations. Cells are incubated for 24 hours at 37°C, 5% CO2. Cell culture supernatants are then harvested and analyzed for IFNβ secretion, using a conventional sandwich ELISA format. Results: Using IFNβ as a readout, we show the ability to activate the STING pathway in both human PBMCs and THP-1 cells, using the STING agonist, 2’3’-cGAMP, as well as its close analogs, 2’3’-cGAM(PS)2 (Rp/Sp) and 2’3’-c-di-AM(PS)2 (Rp/Rp). This assay is robust and capable of measuring the relative potencies of these agonists. Treatment with 2’3’-cGAMP significantly induced IFNβ secretion, in a dose-dependent manner, in both PBMCs and THP-1 cells, with EC50 values of ~70 μM and 124 μM, respectively. Assessment of the potency of different STING agonists in THP-1 cells resulted in EC50 values for 2’3’-cGAM(PS)2 (Rp/Sp) and 2’3’-c-di-AM(PS)2 (Rp/Rp) of 39.7 μM and 10.5 μM, respectively. Induction and measurement of other cytokines, or chemokines, with STING agonists may also be discussed. Conclusions: We have shown the ability to quantitatively measure STING pathway activation in a newly developed in vitro cellular functional assay, using human PBMCs or THP-1 tumor cells. This assay is robust and capable of demonstrating different potencies of different agonists, and thus provides a convenient method to screen for potential therapeutic agents. Citation Format: Satheesh K. Sainathan, Alyssa M. Cracchiolo, Usha Warrior, Alastair J. King. An in vitro cellular functional STING pathway assay to measure the potency of STING agonists, using interferon-β response [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B068.

Inducible MyD88/CD40 (iMC) Costimulation Provides Ligand-Dependent Tumor Eradication By CD123-Specific Chimeric Antigen Receptor T Cells

Introduction: Promising clinical results with CD19-specific chimeric antigen receptor (CAR)-directed T cells for the treatment of B cell leukemia and lymphoma suggest that CARs may be effective in other hematological malignancies, such as acute myeloid leukemia (AML). CD123/IL-3Rα is an attractive CAR-T cell target due to its high expression on both AML blasts and leukemic stem cells (AML-LSCs). However, the antigen is also expressed at lower levels on normal stem cell progenitors presenting a major toxicity concern should CD123-specific CAR-T cells show long-term persistence. Here, we describe a CAR platform, "GoCAR-T", which uses a proliferation-deficient, first generation, CD123-specific CAR together with a ligand (rimiducid (Rim))-dependent costimulatory switch (inducible MyD88/CD40 (iMC)) to provide physician-controlled eradication of CD123+ tumor cells and regulate long-term CAR-T cell engraftment.Methods: T cells were activated with anti-CD3/28 antibodies and subsequently transduced with a bicistronic retrovirus encoding tandem Rim-binding domains (FKBP12v36),cloned in-frame with MyD88 and CD40 cytoplasmic signaling molecules, and first generation CAR targeting CD123 (SFG-iMC-CD123.ζ). The effects ofiMC costimulation on CD123-targeted CARs were assessed in coculture assays with CD123+, EGFPluciferase (EGFPluc)-modified leukemic cell lines (KG1, THP-1 and MOLM-13) with and without Rim using the IncuCyte live cell imaging system. IL-2 production was examined by ELISA from coculture supernatants. In vivo efficacy of iMC-CD123.ζ-modified T cells was assessed using an immune-deficient NSG tumor xenograft model. One million EGFPluc-expressing CD123+ THP-1 tumor cells were injected i.v. into the animals, followed by a single i.v. injection on day 7 with 2.5x106 non-transduced or iMC-CD123.ζ-modified T cells. Groups receiving CAR-T cells subsequently received i.p. injections of Rim (1 mg/kg) or vehicle only on days 0 and 15 post-T cell injection. Animals were evaluated for THP-1-EGFPluc tumor burden and weight change on a weekly basis using IVIS bioluminescent imaging (BLI) and for T cell persistence by flow cytometry and qPCR at day 30 post-T cell injection.Results: SFG-iMC-CD123.ζ efficiently transduced activated T cells (66±8%) and showed antigen-specific cytolytic function against CD123+ leukemic cell lines. However, in coculture assays both CAR antigen recognition and Rim-dependent iMC costimulation were required for IL-2 production (285±41 versus 2,541±255 pg/ml for control and 1 nM Rim, respectively), robust CAR-T cell proliferation (87-fold increase with Rim stimulation) and enhanced tumor cell killing. In NSG mice engrafted with CD123+ THP-1-EGFPluc tumor cells, only animals were treated with iMC-CD123.ζ-modified T cells and 1 mg/kg Rim on days 0 and 15 post-T cell injection controlled tumor growth, showing a 2-log (average radiance: NT = 3.0x108; CAR + Vehicle = 2.4x108; CAR + Rim=8.4x105 p/sec/cm2/sr) reduction in tumor burden compared to mice receiving CAR-T cells and vehicle only (Figure 1). Approximately two weeks after the second Rim injection (day 30), CAR-T cells were infrequent (<1.0%) in the spleen and bone marrow of both CAR groups suggesting that active costimulation is required for CAR-T persistence.Summary: GoCAR-T, a platform comprising a ligand-dependent activation switch and a proliferation-deficient first generation CAR, efficiently eradicates CD123+ leukemic cells when costimulation is provided by systemic rimiducid administration. Deprivation of iMC costimulation results in reduction of CAR-T levels, providing a user-controlled system for managing persistence and safety of CD123-specific CAR-T cells. DisclosuresFoster:Bellicum Pharmaceuticals: Employment. Duong:Bellicum Pharmaceuticals: Employment. Lu:Bellicum Pharmaceuticals: Employment. Chang:Bellicum Pharmaceuticals: Employment. Mahendravada:Bellicum Pharmaceuticals: Employment. Shinners:Bellicum Pharmaceuticals: Employment. Slawin:Bellicum Pharmaceuticals: Employment, Equity Ownership. Bayle:Bellicum Pharmaceuticals: Employment. Spencer:Bellicum Pharmaceuticals: Employment, Equity Ownership.

Triazole tethered C5-curcuminoid-coumarin based molecular hybrids as novel antitubulin agents: Design, synthesis, biological investigation and docking studies

Keeping in view the confines allied with presently accessible antitumor agents and success of C5-curcuminoid based bifunctional hybrids as novel antitubulin agnets, molecular hybrids of C5-curcuminoid and coumarin tethered by triazole ring have been synthesized and investigated for in-vitro cytotoxicity against THP-1, COLO-205, HCT-116 and PC-3 human tumor cell lines. The results revealed that the compounds A-2 to A-9, B-2, B-3, B-7 showed significant cytotoxic potential against THP-1, COLO-205 and HCT-116 cell lines, while the PC-3 cell line among these was found to be almost resistant. Structure activity relationship revealed that the nature of Ring X and the length of carbon-bridge (n) connecting triazole ring with coumarin moiety considerably influence the activity. Methoxy substituted phenyl ring as Ring X and two carbon-bridges were found to be the ideal structural features. The most potent compounds (A-2, A-3 and A-7) were further tested for tubulin polymerization inhibition. Compound A-2 was found to significantly inhibit the tubulin polymerization (IC50 = 0.82 μM in THP-1 tumor cells). The significant cytotoxicity and tubulin polymerization inhibition by A-2 was further rationalized by docking studies where it was docked at the curcumin binding site of tubulin.

The Inhibition of Hsp27 Chaperone Affects the Level of p53 Protein in Tumor Cells

The characteristics of p53 protein content in Jurkat and THP-1 tumor cell line, and mononuclear leukocytes were evaluated from in vitro studies with selective inhibition of the chaperone Hsp27. For the inhibition of Hsp27 KRIBB3 ((5-(5-ethyl-2-hydroxy-4-methoxyphenyl)-4-(4-methoxyphenyl)-isoxasole) was used. The p53 protein concentration was determined by Western blot analysis. The apoptoticly transformed cells with selective inhibitor Hsp27 were assessed by in vitro fluorescence microscopy using FITC-labeled annexin V and propidium iodide. The present study showed that the in vitro inhibition of the chaperone Hsp27 leads to an increase in p53 concentration in tumor cells and a growth of the amount of apoptotic cells in modified Jurkat and THP-1 cultures but revealed no such effects in the mononuclear leukocytes culture. Thus, Hsp27 appeared to play an important regulatory role in the activation of p53 protein of tumor cells.

Effects of HSP27 Chaperone on THP-1 Tumor Cell Apoptosis

The role of Hsp27 (heat shock protein 27) chaperone in regulation of THP-1 tumor cell apoptosis was studied. Realization of tumor cell apoptosis under conditions of in vitro culturing with Hsp27 specific inhibitor (KRIBB3) was evaluated by fluorescent microscopy with FITC-labeled annexin V and propidium iodide. Measurements of Bcl-2 family proteins (Bcl-2, Bax, Bad) in tumor cells incubated with Hsp27 inhibitor were carried out by Western blotting. Chaperone Hsp27 acted as apoptosis inhibitor in THP-1 tumor cells modulating the proportion of antiapoptotic (Bcl-2) and proapoptotic (Bax and Bad) proteins.

Cytotoxic triterpenoids from Abies recurvata

Nine triterpenoids (neoabiestrines A-I, 1-9) including six rearranged lanostanes (1-6) and a rare cycloart-7-ene (7) were isolated from Abies recurvata together with ten known compounds. Their structures were determined by detailed analysis of NMR and MS spectroscopic data. The absolute configurations of 1 and 8 were determined by Cu-Ka X-ray crystallography. Compound 6 showed potent anti-proliferative effect against THP-1 tumor cells with an IC(50) value of 17.8 μg/mL.

Peripheral Substituents of Di(pyridiumyl)porphyrins Affected on Their Interactions with DNA.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Peripheral substituents of di(pyridiumyl)porphyrins affected on their interactions with DNA

meso- and β-Substituted di(pyridiumyl)porphyrins 3, 4, and 7 have been synthesized and their interactions with DNA have been investigated. meso-Substituted porphyrins showed the stronger effect on DNA than that of β-substituted porphyrin. Cytoxicity of compound 3 (IC50) to THP-1 tumor cell was up to 0.11 nM.

Peripheral substituents of di(pyridiumyl)porphyrins affected on their interactions with DNA

meso- and β-Substituted di(pyridiumyl)porphyrins 3, 4, and 7 have been synthesized and their interactions with DNA have been investigated. meso-Substituted porphyrins showed the stronger effect on DNA than that of β-substituted porphyrin. Cytoxicity of compound 3 (IC 50) to THP-1 tumor cell was up to 0.11 nM.