Screening of different drug design tools to predict the mode of action of steroidal derivatives as anti-cancer agents.

There is a pressing need to discover and develop novel drugs against cancer. With the new era of bioinformatics, which integrates different aspects, drug development has been tremendously improved. Recently, extensive research was directed towards the rational modification of steroid molecules against different disease especially cancer. Moreover, heterocyclic steroid derivatives have shown a lot of different biological activities such as antimicrobial, anti-inflammatory, and anti-cancer activities. Molecular docking methods can be used to explore how the steroid derivatives conformations can adopt within the binding sites of specific macromolecular targets involved in cancer progression. We conducted this study to investigate the accuracy of different molecular docking calculations using different steroidal molecular targets, and to define the most accurate one to study the mode of action of steroid derivatives as potential anti-cancer drugs. Our results revealed that the Dock6, PLANTS, AutoDock, GLIDE (SP and XP), and GOLD (ASP, Chemscore, and PLP) software were able to maintain the binding mode of the co-crystallized ligands inside their proteins by achieving RMSD values lower than two. Moreover, molecular docking study revealed that compound 4, and 5 are promising steroidal derivatives as anti-cancer drugs. Further on, the cytotoxic activity of the selected steroidal derivatives were tested against leukemia cell line using MTT assay. The results revealed that compound 4, and 5 were potential cytotoxic agents against THP-1 cells (IC50s were 44.67 µM, and 46.77 µM, respectively), these results are in agreement with the molecular docking study.

AURKA Suppresses Leukemic THP-1 Cell Differentiation through Inhibition of the KDM6B Pathway.

Aberrations in histone modifications are being studied in mixed-lineage leukemia (MLL)-AF9-driven acute myeloid leukemia (AML). In this study, we focused on the regulation of the differentiation of the MLL-AF9 type AML cell line THP-1. We observed that, upon phorbol 12-myristate 13-acetate (PMA) treatment, THP-1 cells differentiated into monocytes by down-regulating Aurora kinase A (AURKA), resulting in a reduction in H3S10 phosphorylation. We revealed that the AURKA inhibitor alisertib accelerates the expression of the H3K27 demethylase KDM6B, thereby dissociating AURKA and YY1 from the KDM6B promoter region. Using Flow cytometry, we found that alisertib induces THP-1 differentiation into monocytes. Furthermore, we found that treatment with the KDM6B inhibitor GSK-J4 perturbed the PMA-mediated differentiation of THP-1 cells. Thus, we discovered the mechanism of AURKA-KDM6B signaling that controls the differentiation of THP-1 cells, which has implications for biotherapy for leukemia.

Bioassay-Guided Fractionation and In Vitro Antiproliferative Effects of Fractions of Artemisia nilagirica on THP-1 cell line.

ABSTACT Artemisia nilagirica (Clarke) is a widely used medicinal herb in Indian traditional system of medicine. Therefore, the present study was designed to evaluate the effects of A. nilagirica extracts/fractions on inhibition of proliferation and apoptosis in a human monocytic leukemia (THP-1) cell line. The crude extracts (A. nilagirica ethyl acetate extract [ANE] and A. nilagirica methanolic extract [ANA]) showed cytotoxic activity toward THP-1 cells with the IC50 values of 38.21 ± 7.37 and 132.41 ± 7.19 µg/ml, respectively. However, the cytotoxic activity of active fractions (ANE-B and ANM-9) obtained after column chromatography was found to be much more pronounced than their parent extracts. The IC50 values of ANE-B and ANM-9 were found to be 27.04 ± 2.54 µg/ml and 12.70 ± 4.79 µg/ml, respectively, suggesting greater susceptibility of the malignant cells. Cell cycle analysis and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end-labeling (TUNEL) assay revealed that inhibition of cell growth by A. nilagirica fractions on THP-1 cells was mediated by apoptosis. Active fractions of A. nilagirica increased the expression levels of caspase-3, -7, and poly-ADP-ribose polymerase (PARP), a critical member of the apoptotic pathway. These results suggested that active fractions of A. nilagirica may play a promising role in growth suppression by inducing apoptosis in human monocytic leukemic cells via mitochondria-dependent and death receptor-dependent apoptotic pathways.

Effects of globularifolin on cell survival, nuclear factor-κB activity, neopterin production, tryptophan breakdown and free radicals in vitro.

The potential effects of globularifolin, an acylated iridoid glucoside, on cell survival, inflammation markers and free radicals scavenging were investigated. Viability assay on human myelomomonocytic cell line THP-1 and human peripheral blood mononuclear cells (PBMC) using the Cell-Titer Blue assay proved that globularifolin had no toxic effect at the tested concentrations. Conversely, it is proportional to the dose globularifolin increased growth of THP-1 cells (p <0.01). On human PBMC, globularifolin at 6.25 and 12.5 µM concentrations showed a stimulatory effect, while at 12.5-200 µM it suppressed response of PBMC to stimulation with phytohemagglutinin (PHA). Globularifolin (50-200 µM) enhanced neopterin formation dose-dependently, whereas tryptophan breakdown was not influenced. At 50-200 µM in unstimulated PBMC in THP-1 cells, globularifolin induced a significant expression of nuclear factor-κB (NF-κB) as was quantified by Quanti-Blue assay. By contrast, in lipopolysaccharide (LPS)-stimulated cells, the higher concentrations of globularifolin suppressed NF-κB expression dose-dependently and a significant decrease was observed at 200 µM concentration. A positive correlation was found between increased neopterin and NF-κB activity (p <0.01). Similarly, a positive correlation was observed between neopterin levels in mitogen-induced cells and NF-κB activity in LPS-stimulated cells after treatment with globularifolin (p=0.001). The free radical scavenging capacity of globularifolin evaluated by Oxygen Radical Absorbance Capacity (ORAC) assay showed relative ORAC values of 0.36±0.05 µmol Trolox equivalent/µmol. All together, results show that natural antioxidant globularifolin might represent a potential immunomodulatory as well as proliferative agent, which deserves further in vitro and in vivo studies.

Curcumin induces the apoptosis of human monocytic leukemia THP-1 cells via the activation of JNK/ERK pathways.

BACKGROUND: Curcumin is a principal compound of turmeric, commonly used to treat tumors and other diseases. However, its anti-cancer activity in human acute monocytic leukemia THP-1 cells is not clear. This study aimed to study the anti-cancer effect and action of curcumin on THP-1 cells. METHODS: THP-1 parental cells and PMA-treated THP-1 cells, were used as in vitro models to evaluate the anti-cancer effect and mechanism of curcumin. Apoptosis and its mechanism were evaluated by WST-1, flow cytometry and Western blotting. MAPK inhibitors were used to further confirm the molecular mechanism of curcumin-induced THP-1 cell apoptosis. RESULTS: Curcumin induced cell apoptosis of THP-1 cells as shown by cell viability, cell cycle analysis and caspase activity. Curcumin significantly increased the phosphorylation of ERK, JNK and their downstream molecules (c-Jun and Jun B). Inhibitor of JNK and ERK reduced the pro-apoptotic effect of curcumin on THP-1 cells as evidenced by caspase activity and the activation of ERK/JNK/Jun cascades. On the contrary, the pro-apoptotic effect of curcumin was abolished in the differentiated THP-1 cells mediated by PMA. CONCLUSIONS: This study demonstrates that curcumin can induce the THP-1 cell apoptosis through the activation of JNK/ERK/AP1 pathways. Besides, our data suggest its novel use as an anti-tumor agent in acute monocytic leukemia.

Capsaicin attenuates palmitate-induced expression of macrophage inflammatory protein 1 and interleukin 8 by increasing palmitate oxidation and reducing c-Jun activation in THP-1 (human acute monocytic leukemia cell) cells.

Capsaicin, a spicy component of hot peppers, has been shown to improve inflammatory disease and obesity. In this study, we tested the hypothesis that the anti-inflammatory activity of capsaicin can be used to improve free fatty acid (FFA)-induced inflammation by reducing gene expression of macrophage inflammatory protein 1 (MIP-1) and interleukin 8 (IL-8) in THP-1 (human acute monocytic leukemia cell) macrophages. To investigate whether capsaicin ameliorates palmitate-induced MIP-1 and IL-8 gene expressions, we treated THP-1 cells with palmitate in the presence or absence of capsaicin and measured MIP-1 and IL-8 by real-time polymerase chain reaction. To elucidate the mechanism by which capsaicin effects on palmitate-induced MIP-1 and IL-8 gene expressions, we performed immunoblotting with stress kinase-related antibodies and measured palmitate oxidation and palmitate oxidation-related gene expression. Palmitate and stearate but not the unsaturated FFA oleate significantly increased MIP-1 and IL-8 expressions in THP-1 macrophages. Treatment with capsaicin or FFA oxidation stimulators inhibited palmitate-induced MIP-1 and IL-8 expressions in THP-1 macrophages. Capsaicin increased the gene expression of carnitine palmitoyltransferase 1 and the ß-oxidation of palmitate. Furthermore, capsaicin significantly reduced palmitate-stimulated activation of c-Jun N-terminal kinase, c-Jun, and p38. Our data suggest that the attenuation of palmitate-induced MIP-1 and IL-8 gene expressions by capsaicin is associated with reduced activation of c-Jun N-terminal kinase, c-Jun, and p38 and preserved ß-oxidation activity.

Ginsenoside Rh1 inhibits the invasion and migration of THP-1 acute monocytic leukemia cells via inactivation of the MAPK signaling pathway.

Ginsenoside Rh1 has been reported to possess antiallergic and anti-inflammatory activities, but its effects on monocytes remain to be determined. Herein, we investigated the effects of Rh1 on the expression of MCP-1 and CCR2, activation of MAPK signaling, and chemotaxis of monocytes. Treatment of Rh1 decreased the levels of MCP-1 and CCR2 and the expression of VLA5 and activated ß1 integrin on the cell surface, and attenuated the phosphorylation of MAPKs. Based on these results, the inhibitory effects of Rh1 on monocyte function should be regarded as a promising new anti-inflammatory response with a potential therapeutic role against inflammation-dependent diseases.

Ganoderma lucidum polysaccharides in human monocytic leukemia cells: from gene expression to network construction.

BACKGROUND: Ganoderma lucidum has been widely used as a herbal medicine for promoting health and longevity in China and other Asian countries. Polysaccharide extracts from Ganoderma lucidum have been reported to exhibit immuno-modulating and anti-tumor activities. In previous studies, F3, the active component of the polysaccharide extract, was found to activate various cytokines such as IL-1, IL-6, IL-12, and TNF-alpha. This gave rise to our investigation on how F3 stimulates immuno-modulating or anti-tumor effects in human leukemia THP-1 cells. RESULTS: Here, we integrated time-course DNA microarray analysis, quantitative PCR assays, and bioinformatics methods to study the F3-induced effects in THP-1 cells. Significantly disturbed pathways induced by F3 were identified with statistical analysis on microarray data. The apoptosis induction through the DR3 and DR4/5 death receptors was found to be one of the most significant pathways and play a key role in THP-1 cells after F3 treatment. Based on time-course gene expression measurements of the identified pathway, we reconstructed a plausible regulatory network of the involved genes using reverse-engineering computational approach. CONCLUSION: Our results showed that F3 may induce death receptor ligands to initiate signaling via receptor oligomerization, recruitment of specialized adaptor proteins and activation of caspase cascades.

Apoptosis inducing activity of viscin, a lipophilic extract from Viscum album L.

Detection of antiproliferative activity and bioactivity-guided fractionation of viscin, a lipophilic extract from Viscum album L., led to the isolation of betulinic acid, oleanolic acid and ursolic acid as active components. Viscin, betulinic acid, oleanolic acid and ursolic acid inhibited growth and induced apoptotic cell death in Molt4, K562 and U937 leukaemia cells. The growth inhibitory effect of viscin was more pronounced in Molt4 and U937 cells (IC50 (concentration that inhibited cell proliferation by 50%): 118 +/- 24 and 138 +/- 24 microg mL(-1)) than in K562 cells (IC50: 252 +/- 37 microg mL(-1)). Oleanolic acid was the least effective in all cell lines (7.5-45.5% inhibition at 10 microg mL(-1)) and ursolic acid the most active in Molt4 and U937 cells (81.8 and 97.8% inhibition, respectively, at 5 microg mL(-1)). A dose-dependent loss of membrane phospholipid asymmetry associated with apoptosis was induced in all cell lines as shown in flow cytometry by the externalization of phosphatidylserine and morphological changes in cell size and granularity. There were differences in individual cell lines' response towards the apoptosis-inducing effect of viscin, betulinic acid, oleanolic acid and ursolic acid. The triterpenoids beta-amyrin, beta-amyrinacetate, lupeol, lupeolacetate, beta-sitosterol and stigmasterol, and the fatty acids oleic acid, linoleic acid, palmitic acid and stearic acid were also present in the lipophilic extract.

Identification of 6-methylsulfinylhexyl isothiocyanate as an apoptosis-inducing component in wasabi.

The ethanol extract from Japanese horseradish wasabi was found to inhibit cell proliferation in human monoblastic leukemia U937 cells by inducing apoptotic cell death. Separation by methods including silica gel chromatography and preparative HPLC gave an active compound, which was identified as 6-methylsulfinylhexyl isothiocyanate (6-HITC). Several lines of evidence indicated that 6-HITC induced apoptosis in U937 cells and human stomach cancer MKN45 cells. Thus, 6-HITC is potentially useful as a natural anti-cancer agent.

Roles of extracellular signal-regulated kinase and p38 mitogen-activated protein kinase in apoptosis of human monoblastic leukemia U937 cells by lectin-II isolated from Korean mistletoe.

The mitogen-activated protein kinase (MAPK) family members have been implicated in cell survival. We have previously demonstrated that cytotoxic lectin-II isolated from Korean mistletoe induces apoptotic cell death in the human monoblastic leukemia cell line, U937, via the activation of the stress-activated protein kinases/c-Jun N-terminal kinase (SAPK/JNK). In the present study, the roles of extracellular signal-regulated kinases (ERK1/2) and p38 MAPK in lectin-II-induced apoptosis have been investigated. Treatment of U937 cells with lectin-II resulted in apoptotic DNA fragmentation, which was preceded by the activation of ERK1/2, p38 MAPK and SAPK/JNK. This lectin-II-induced DNA fragmentation was significantly enhanced when ERK1/2 activation was selectively inhibited by PD098059. 12-O-tetradecanoylphorbol-13-acetate, which stimulates ERK activity in U937 cells, markedly reduced lectin-II-induced DNA fragmentation. Inhibition of p38 MAPK activity with p38-specific inhibitor, SB203580, partially inhibited lectin-II-induced DNA fragmentation. These results suggest that ERK1/2 and p38 MAPK may have opposite effects on cell survival in response to cytotoxic mistletoe lectin-II, which may contribute to the modulation of lectin-II-mediated cytotoxic activity.

t(11;14)(q23;q24) generates an MLL-human gephyrin fusion gene along with a de facto truncated MLL in acute monoblastic leukemia.

More than 20 different partner genes with MLL have been cloned from leukemia cells with translocations involving chromosome 11 band q23 (11q23). All reported partner genes fused in-frame to MLL and the fusion cDNA encode chimeric MLL proteins with a significant portion derived from the partner genes. We analyzed one patient with de novo acute monoblastic leukemia with t(11;14)(q23;q24) and identified that a human homologue of gephyrin (human gephyrin) fused with MLL. Gephyrin is a rat glycine receptor-associated protein, which forms submembranous complexes and anchor glycine or gamma-aminobutyric acidA receptors to microtubules. Alternative splicing of human gephyrin gene created two different forms of fusion cDNA. In one form, human gephyrin gene fused in-frame to MLL exon 9, and the chimeric product had COOH terminus of human gephyrin protein, including the tubulin binding site. In the other, the reading frame terminated shortly after the fusion point. As a result, only seven amino acids with no known function were attached to the NH2 terminus of MLL protein. The functional significance of this de facto truncated MLL gene product is not clear.

Relationship between TNF-alpha and iron metabolism in differentiating human monocytic THP-1 cells.

The human monocytic cell line THP-1 differentiates along the macrophage line after phorbol-12-myristate-13-acetate (PMA) supplementation and can be stimulated to secrete tumour necrosis factor alpha (TNF-alpha) by interferon gamma (IFN-gamma) addition. We found that, in the early stage of differentiation (1-48 h), PMA induction elicited an upregulation of intracellular H ferritin and H ferritin binding sites and a downregulation of transferrin receptor. In addition, we found that iron administration to PMA-differentiating cells induced the expression of TNF-alpha mRNA and TNF-alpha secretion to levels even higher than those induced by IFN-gamma alone. The iron chelator desferrioxamine showed the opposite effect and reduced TNF-alpha release. In contrast, preincubation of the cells with iron before PMA induction resulted in a decrease of the TNF-alpha secretion induced by IFN-gamma, whereas the opposite was true after preincubation with desferrioxamine. The data support a co-ordinate interaction between iron and TNF-alpha in monocyte macrophages, with an iron-mediated upregulation of TNF-alpha in the early phase of differentiation and an iron-mediated inhibition at later stages. This complex relationship has to be considered in evaluating the effects of iron on inflammation.

Human monoblastoid cell line U-937 cultured in protein-free medium: immunophenotype, cytochemical and biochemical markers.

Human monoblastoid cell line U-937 was adapted to grow in protein-free (protein-free hybridoma--PFH) medium and cloned by limiting dilution. Resulting cell subline (U-937/PF) cultured in protein-free medium was characterized by immunological, cytochemical and biochemical techniques. There were no major differences in immunophenotype (determined by FACS analysis with monoclonal antibodies directed to HLA and CD antigens) and cytochemical markers between the U-937/PF cells cultured in protein-free cell culture medium and parental U-937 cell cultured in serum-supplemented medium. Maximal cell density was slightly decreased in protein-free culture as compared to the parental cell line in FCS-supplemented medium. Cell viability and cell DNA histograms (determined by propidium iodide cytofluorimetry) showed no major differences between parental U-937 and U-937/PF cells. Phorbol ester (TPA)-induction of differentiation-associated cell markers resulted in a proliferation arrest and accumulation of G0/G1 cells in both sublines. All-trans retinoic acid and, to a lesser extent, TPA-stimulated NBT reduction was higher in parental U-937 cells cultured in serum-supplemented medium as compared to U-937/PF cells. Quantitative differences in the expression and inducibility of some cytochemical markers (beta-glucuronidase, chloroacetate esterase) were found between both examined sublines. Described U-937/PF subline cultured in a protein-free cell culture medium (PFH) appeared as a potential tool for studies of in vitro inducing agents and serum components with differentiation promoting (or inhibiting) activities.