Human Leukemia Cell Line U937 Research Articles

Induction of apoptosis by piceatannol in human leukemic U937 cells through down-regulation of Bcl-2 and activation of caspases

Piceatannol is a polyphenol that is found in abundant quantities in grapes and wine. Although recent experimental data revealed the proapoptotic potency of piceatannol, the molecular mechanisms underlying the anti-leukemic activity have not yet been studied in detail. This study examined the effects of piceatannol on the growth of the human leukemia cell line U937. The results showed that piceatannol inhibits the viability of U937 cells by inducing apoptosis, as evidenced by the formation of apoptotic bodies, DNA fragmentation and the accumulation of the sub-G1 phase. RT-PCR and immunoblotting data showed that treating the cells with piceatannol caused the down-regulation of anti-apoptotic Bcl-2 and cIAP-2 expression. Piceatannol-induced apoptosis was also associated with the proteolytic activation of caspase-3, and the degradation/cleavage of poly (ADP-ribose) polymerase protein. z-DEVD-fmk, a caspase-3-specific inhibitor, blocked the activation of caspase-3 and increased the survival of the piceatannol-treated U937 cells, suggesting that caspase-3 activation is essential for piceatannol-induced apoptosis.

Epigenetic Multiple Ligands: Mixed Histone/Protein Methyltransferase, Acetyltransferase, and Class III Deacetylase (Sirtuin) Inhibitors

A number of new compounds bearing two ortho-bromo- and ortho, ortho-dibromophenol moieties linked through a saturated/unsaturated, linear/(poly)cyclic spacer (compounds 1- 9) were prepared as simplified analogues of AMI-5 (eosin), a recently reported inhibitor of both protein arginine and histone lysine methyltransferases (PRMTs and HKMTs). Such compounds were tested against a panel of PRMTs (RmtA, PRMT1, and CARM1) and against human SET7 (a HKMT), using histone and nonhistone proteins as a substrate. They were also screened against HAT and SIRTs, because they are structurally related to some HAT and/or SIRT modulators. From the inhibitory data, some of tested compounds ( 1b, 1c, 4b, 4f, 4j, 4l, 7b, and 7f) were able to inhibit PRMTs, HKMT, HAT, and SIRTs with similar potency, thus behaving as multiple ligands for these epigenetic targets (epi-MLs). When tested on the human leukemia U937 cell line, the epi-MLs induced high apoptosis levels [i.e., 40.7% ( 4l) and 42.6% ( 7b)] and/or massive, dose-dependent cytodifferentiation [i.e., 95.2% ( 1c) and 96.1% ( 4j)], whereas the single-target inhibitors eosin, curcumin, and sirtinol were ineffective or showed a weak effect.

A factor inducing differentiation of the human monocytic cell line U-937 produced by 12-O-tetradecanoylphorbol 13-acetate-treated U-937.

A new factor capable of inducing differentiation of human leukemic cell line U-937 into macrophages was found and partially purified from the conditioned medium of U-937 previously treated with 12-O-tetradecanoylphorbol 13-acetate. The purification procedure included ultrafiltration, DEAE-Sephacel and butyl-Toyopearl column chromatography. The purified factor gave a major band of protein with a molecular weight of 67,000 daltons which coincided with the biological activity of differentiation-inducing factor, and it was not adsorbed on a concanavalin A column. These results suggest that this factor is distinct from other differentiation-inducing factors.

The Interferon Consensus Sequence-binding Protein (ICSBP/IRF8) Represses PTPN13 Gene Transcription in Differentiating Myeloid Cells

The interferon consensus sequence-binding protein (ICSBP/IRF8) is an interferon regulatory factor that is expressed in myeloid and B-cells. ICSBP-deficient mice develop a myeloproliferative disorder characterized by cytokine hypersensitivity and apoptosis resistance. To identify ICSBP target genes involved in these effects, we screened a CpG island microarray with chromatin that co-immunoprecipitated with ICSBP from myeloid cells. Using this technique, we identified PTPN13 as an ICSBP target gene. PTPN13 encodes Fas-associated phosphatase 1 (Fap-1), a ubiquitously expressed protein-tyrosine phosphatase. This was of interest because interaction of Fap-1 with Fas results in Fas dephosphorylation and inhibition of Fas-induced apoptosis. In this study, we found that ICSBP influenced Fas-induced apoptosis in a Fap-1-dependent manner. We also found that ICSBP interacted with a cis element in the proximal PTPN13 promoter and repressed transcription. This interaction increased during myeloid differentiation and was regulated by phosphorylation of conserved tyrosine residues in the interferon regulatory factor domain of ICSBP. ICSBP deficiency was present in human myeloid malignancies, including chronic myeloid leukemia. Therefore, these studies identified a mechanism for increased survival of mature myeloid cells in the ICSBP-deficient murine model and in human myeloid malignancies with decreased ICSBP expression.

Synthesis of New Pyrrolo[1,2-a]quinoxaline Derivatives as Potential Inhibitors of Akt Kinase

Akt kinases are attractive targets for small molecule drug discovery because of their key role in tumor cell survival/proliferation and their overexpression/activation in many human cancers. Recent efforts in the development and biological evaluation of small molecule inhibitors of Akt have led to the identification of novel Akt kinase inhibitors, based on a quinoxaline or pyrazinone scaffold. A series of new substituted pyrrolo[1,2-a]quinoxaline derivatives, structural analogues of these active quinoxaline or pyrazinone pharmacophores, was synthesized from various substituted 2-nitroanilines or 1,2-phenylenediamine via multistep heterocyclization process. These new compounds were tested for their in vitro ability to inhibit the proliferation of the human leukemic cell lines K562, U937 and HL60, and the breast cancer cell line MCF7. Three of these human cell lines (K562, U937 and MCF7) exhibited an active phosphorylated Akt form. The most promising active pyrroloquinoxalines were found to be 1a that inhibited K562 cell line proliferation with an IC50 of 4.5 μM, and 1h that inhibited U937 and MCF7 cell lines with IC50 of 5 and 8 μM, respectively. These two candidates exhibited more potent activities than the reference inhibitor A6730.

Antitumor effect of interferon-on U937 human acute leukemia cells in vitro and its molecular mechanism

In order to investigate the antitumor effect and molecular mechanism of interferon-alpha (IFN-alpha) on human acute myeloid leukemia cell line U937 cells in vitro, the proliferation of U937 cells was determined by MTT assay, the apoptosis rate was analyzed by flow cytometry (FCM), and the mRNA expression of cell cycle regulatory protein cyclin E was detected by RT-PCR. The results showed that IFN-alpha could inhibit the proliferation of U937 cells significantly in a dose-and time-dependent way (P<0.01), and induce the apoptosis of U937 cells also in a dose-and time-dependent manner at the concentration of 1000-4000 U/L (P<0.01). The apoptosis rate of U937 cells was even over 50% when cultured with IFN-alpha for 36-48 h at the concentration of 2000-4000 U/L. Moreover, the expression of cyclin E mRNA was markedly inhibited by the addition of IFN-alpha, and the inhibition was time-dependent (P<0.01). It was concluded that the anti-leukemia mechanism of IFN-alpha might be correlated with its antiproliferative and apoptotic inducing effects, and the down-regulation of the cyclin E expression might be one of its molecular mechanisms.

Synthesis, characterization, crystal structure and antiproliferative in vitro activity of long-chain aliphatic thiosemicarbazones and their Ni(II) complexes

In the course of a research on metal-based compounds active on white blood cell cancers (leukemia, lymphoma and myeloma) nine aliphatic thiosemicarbazones and their nickel complexes have been synthesized with the aim to test their effect on histiocytic lymphoma U937 cell proliferation. All compounds were characterized by elemental, IR and NMR spectra analyses and for five of the complexes also by X-ray crystallography diffraction analysis. Heptanal thiosemicarbazone and the corresponding nickel complex were chosen after a solubility/stability test to carry out preliminary experiments in vitro on human leukemia cell line U937. The complex possesses a remarkable biological activity in inhibiting cell growth and in inducing apoptosis.

Selective guanosine binding and cytotoxicity of a benzimidazole derived dinickel complex

A water-soluble dinickel(II) complex of ethylene glycol-bis(β-aminoethyl ether) N,N,N′,N′-tetrakis(2-benzimidazoyl) (EGTB) was synthesized and fully characterized. The complex crystallizes in a monoclinic system with space group P21/c, a=10.125(1)Å, b=28.393(3)Å, c=11.026(1)Å, and β=98.966(2)°. The hexa-coordinated nickel(II) centers in the centrosymmetric complex adopt a distorted octahedron geometry. The complex binds to purine nucleotides covalently and shows a clear preference for guanosine-5′-monophosphate (5′-GMP) over adenosine-5′-monophosphate (5′-AMP). Its binding to calf thymus DNA (CT-DNA) induces a remarkable conformational variation. The cytotoxic activity of the complex was tested against diverse cell lines including human leukemic cell line U937, macrophage cell line Raw 264.7, human cervical cancer cell line Hela, and human hepatocytes cell line L02. The complex shows a significant inhibition against U937 and Raw 264.7 but little inhibition against Hela and L02.

Bispyridinium Dienes: Histone Deacetylase Inhibitors with Selective Activities

A novel synthetic route to the cyclostellettamines 1 using as the key step a microwave-mediated macrocyclic ring-closing metathesis of precursors bispyridinium dienes 10 followed by catalytic hydrogenation has been developed. The open-chain bispyridinium dienes 10 showed uniformly higher histone deacetylase (HDAC) inhibitory potency than the natural products. Diene 10b inhibited HDAC1 and was inactive on HDAC4, whereas 10a showed a weak inhibition of HDAC1 and a higher activity on HDAC4. Neither 10b nor 10a inhibited isoforms HDAC2 and HDAC3. Cell cycle analysis, cell differentiation, and apoptosis as well as evaluation of the acetylation status of H3 lysine tails, up-regulation of p21WAF1/CIP1, and alpha-tubulin acetylation induced by the dienes 10 and cyclostellettamines 1 were also carried out on the human leukemia U937 cell line. These enzymatic and functional assays suggest that 10b is a HDAC1-selective inhibitor and 10a is a HDAC subclass IIa-selective inhibitor.

Novel pyrrole-containing histone deacetylase inhibitors endowed with cytodifferentiation activity

A novel series of aroyl-pyrrolyl-hydroxy-amides (APHAs) active as histone deacetylase (HDAC) inhibitors has been reported. The new derivatives were designed by replacing the benzene ring of the prototype 1 with both aromatic and aliphatic, monocyclic and polycyclic rings (compounds 3a–i), or by inserting a number of substituents on the methylene linker of 1 (compounds 4a–l). Compounds 3a–i and 4a–l were active at sub-micromolar level against the maize deacetylases HD1-B (class I), HD1-A (class II), and HD2. Tested at 5μM against human HDAC1 and HDAC4, 3b, 4a, and 4j showed significant HDAC1 inhibition, whereas on HDAC4 only 4a was highly effective. On the human leukemia U937 cell line, the same compounds did not alter the cell cycle phases and failed in inducing apoptosis. However, they displayed granulocytic differentiation at 5μM, with 3b being the most potent (76% CD11c positive cells). Tested to evaluate their effects on histone H3 and α-tubulin acetylation, 3b and 4a showed high H3 acetylation, whereas 4a and 4b were the most potent with α-tubulin as a substrate.

D-3-deoxy-dioctanoyl-PI induces cell death in the human leukemic monocyte lymphoma cell line U-937 by disruption of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway (89.33)

Abstract D-3-deoxy-phosphatidylinositol derivatives have a potentially wide array of targets in the phosphatidylinositol 3-kinase (PI3K)/Akt signalling pathway. As such, these phosphatidylinositol molecules have been shown to have cytotoxic activity against a variety of human cancer cell lines. Here, we demonstrate that the D-3-deoxy-dioctanoyl-PI (D-diC8PI), but not the L-isomer or D-3,5-dideoxy-diC8PI, is able to induce the death of the U-937 human leukemic monocyte lymphoma cell line, even at low concentrations (&amp;lt;50 μM). In order to further investigate the mechanism of the cytotoxicity of D-3-deoxy-diC8PI we examined its effect on the activity of various molecules in the PI3K/Akt pathway. In vitro, low concentrations (&amp;lt;50 μM) of D-3-deoxy-diC8PI have no significant effect on the activity of PI3K or PTEN. However, incubation of U-937 cells with D-3-deoxy-diC8PI at these concentrations results in significant decreases in phosphorylation of key components of the PI3K/Akt signalling pathway, including the transcription initiation factor eIF4e and the translation factor S6 ribosomal protein. These data further delineate the mechanism of action of such cytotoxic D-3-deoxy-PI compounds.

MJ‐29 interacts with microtubule polymerization and induces mitotic arrest following apoptosis in human leukemia U937 cell line

MJ‐29 interacts with microtubule polymerization and induces mitotic arrest following apoptosis in human leukemia U937 cell line

갈파래(Ulva lactuca) 추출분획의 암 세포주에 대한 세포독성 및 면역활성 효과

본 연구는 갈파래(Ulva lactuca) 추출분획의 암세포주에 대한 세포독성 및 면역활성 효과를 조사하였다. 갈파래의 에탄올 추출물로부터 분획물의 제조는 hexane, ethyl ether, methanol, butanol, <TEX>$H_2O$</TEX>의 용매를 이용하여 행하였다. 인간 백혈암세포주(U937), 생쥐 신경아종세포주(NB41A3), 인체간암세포주(HepG2),큰지 신경교세포주(C6) 등에 대한 갈파래 분획물의 세포독성을 측정하였다. 갈파래의 Ethyl ether 층은 4종류의 세포 모두에서 가장 높은 세포독성을 나타냈다. 또한 수층 역시 비교적 높은 세포독성을 나타냈다. 4종류의 세포 모두에서 농도의존적 경향을 나타냈다. 갈파래 분획물의 큰쥐 대식세포주(RAW 264.7)에 대한 면역활성 효과도 조사하였다. 갈파래 추출물의 5가지 분획물 모두에서 농도의존적으로 NO 생산을 활성화시켰다. 이러한 결과들로 잔파래가 항암 덴 면역활성을 나타내는 천연 소재개발에 있어 유용한 후보가 될 수 있을 것으로 사료된다. Extracted fractions of the green seaweed Ulva lactuca were studied to verify the cytotoxicity and immunostimulating activity. The fractions from the ethanol extract of U. lactuca were prepared by the systematic extraction procedure with solvents such as hexane, ethyl ether, methanol, butanol and <TEX>$H_2O$</TEX>. The cytotoxic effects of U. lactuca fractions against human leukemia cell line U937, mouse neuroblastoma cell line (NB41A3), human hepatoma cell line (HepG2) and rat glioma cell line (C6) were investigated. Ethyl ether fraction showed the highest cytotoxicity against all four cell lines tested. In addition, <TEX>$H_2O$</TEX> fraction also showed relatively high cytotoxicity. Dose dependent patterns were observed on all four cell lines. The immune-stimulating effects of U. lactuca fractions on rat macrophage cell line (RAW 264.7) were also investigated. All five fractions of U. lactuca extract stimulated NO production with concentration dependant manner. These results suggest that U. lactuca may be a useful candidate for a natural cancer preventing and immune-stimulating agents.

Small-Molecule Inhibitors of Histone Acetyltransferase Activity: Identification and Biological Properties

Starting from a yeast phenotypic screening performed on 21 compounds, we described the identification of two small molecules (9 and 18) able to significantly reduce the S. cerevisiae cell growth, thus miming the effect of GCN5 deletion mutant. Tested on a GCN5-dependent gene transcription assay, compounds 9 and 18 gave a high reduction of the reporter activity. In S. cerevisiae histone H3 terminal tails assay, the H3 acetylation levels were highly reduced by treatment with 0.6-1 mM 9, while 18 was effective only at 1.5 mM. In human leukemia U937 cell line, at 1 mM 9 and 18 showed effects on cell cycle (arrest in G1 phase, 9), apoptosis (9), and granulocytic differentiation (18). When tested on U937 cell nuclear extracts to evaluate their histone acetyltransferase (HAT) inhibitory action, both compounds were able to reduce the enzyme activity when used at 500 microM. Another quinoline, compound 22, was synthesized with the aim to improve the activity observed with 9 and 18. Tested in the HAT assay, 22 was able to reduce the HAT catalytic action at 50 and 25 microM, thereby being comparable to anacardic acid, curcumin, and MB-3 used as references. Finally, in U937 cells, compounds 9 and 18 used at 2.5 mM were able to reduce the extent of the acetylation levels of histone H3 (9) and alpha-tubulin (9 and 18). In the same assay, 22 at lower concentration (100 microM) showed the same hypoacetylating effects with both histone and non-histone substrates.

Atmospheric levels and cytotoxicity of PAHs and heavy metals in TSP and PM 2.5 at an electronic waste recycling site in southeast China

Twenty-nine air samples of total suspended particles (TSP, particles less than 30–60 μm) and thirty samples of particles with aerodynamic diameter smaller than 2.5 μm (PM 2.5) were collected at Guiyu, an electronic waste (e-waste) recycling site in southeast China from 16 August 2004 to 17 September 2004. The results showed that mass concentrations contained in TSP and PM 2.5 were 124±44.1 and 62.12±20.5 μg m −3, respectively. The total sum of 16 USEPA priority polycyclic aromatic hydrocarbons (PAHs) associated with TSP and PM 2.5 ranged from 40.0 to 347 and 22.7 to 263 ng m −3, respectively. Five-ring and six-ring PAHs accounted for 73% of total PAHs. The average concentration of benzo(a) pyrene was 2–6 times higher than in other Asian cities. Concentrations of Cr, Cu and Zn in PM 2.5 of Guiyu were 4–33 times higher than in other Asian countries. In general, there were significant correlations between concentrations of individual contaminants in TSP with PM 2.5 (i.e. PAHs, Cd, Cr, Cu, Pb, Zn, Mn except Ni and As). The high concentrations of both PAHs and heavy metals in air of Guiyu may impose a serious environmental and health concern. Cytotoxicity of the extract of TSP and PM 2.5 of ten 24 h samples collected against human promonocytic leukemia cell line U937 (ATCC 1593.2) was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cytotoxicity assay. The results showed that under the same concentrations of extract, PM 2.5 cytotoxicity was 2–4 times higher than TSP.

Hypericin‐induced Photocytotoxicity is Connected with G2/M Arrest in HT‐29 and S‐Phase Arrest in U937 Cells

Susceptibility of the HT-29 human colon adenocarcinoma cell line and human myeloid leukemia cell line U937 to hypericin-mediated photocytotoxicity was investigated and compared in this study. Cellular parameters as viability, cell number, metabolic activity and total protein amount were monitored in screening experiments with subsequent cell-cycle analysis and apoptosis detection to determine the cellular response of the different tumor types to various concentrations of photoactivated hypericin. The results show concentration dependence of the photosensitizer's cytotoxicity on the studied cell lines, with higher sensitivity of U937 cells. Whereas the two extreme hypericin concentrations (1 x 10(-9) M and 1 x 10(-6) M) resulted in similar changes in all tested cellular parameters on the two studied cell lines, 1 x 10(-8) M and 1 x 10(-7) M hypericin treatment resulted in different responses of the cell lines in all monitored parameters except for viability. Although leukemic cells proved sensitive to both 1 x 10(-8) M and 1 x 10(-7) M hypericin, significant changes on HT-29 cells were detected only after the 1 x 10(-7) M hypericin concentration. Cell-cycle arrest was related to simultaneously occurring apoptosis in colon cancer. Remarkable is the difference in cell-cycle profile where G2/M arrest in colon cancer cells versus accumulation of leukemic cells in the S phase appears. This suggests that hypericin treatment affecting the cell-cycle machinery of different cancer cells is not universal in effect.

Indian black scorpion ( Heterometrus bengalensis Koch) venom induced antiproliferative and apoptogenic activity against human leukemic cell lines U937 and K562

Venoms are rich source of several bioactive compounds that possess therapeutic potentials. The different constituents of scorpion venom can modulate cell proliferation, cell growth and cell cycle. In the present communication, the cytotoxic activity of Indian black scorpion ( Heterometrus bengalensis) venom was explored on human leukemic U937 and K562 cells. Scorpion venom induced U937 and K562 cell growth inhibition and the IC 50 value calculated to be 41.5 μg/ml (U937) and 88.3 μg/ml (K562). The scorpion venom showed characteristic features of apoptosis such as membrane blebbing, chromatin condensation and DNA degradation in both the cells as evidenced by confocal, fluorescence, scanning electron microscopy. Scorpion venom (IC 50 dose, 48 h) induced DNA fragmentation as evidenced by comet formation. Flow-cytometric assay revealed a significant amount of apoptotic cells (early and late) due to scorpion venom treatment. The venom induced cell cycle arrest was observed with maximum cell accumulation at sub-G 1 phase. Thus, the Indian scorpion ( H. bengalensis) venom possessed antiproliferative, cytotoxic and apoptogenic activity against human leukemic cells.

Identification of Proteins Cleaved Downstream of Caspase Activation in Monocytes Undergoing Macrophage Differentiation

We have shown previously that caspases were specifically involved in the differentiation of peripheral blood monocytes into macrophages while not required for monocyte differentiation into dendritic cells. To identify caspase targets in monocytes undergoing macrophagic differentiation, we used the human monocytic leukemic cell line U937, whose macrophagic differentiation induced by exposure to 12-O-tetradecanoylphorbol 13-acetate (TPA) can be prevented by expression of the baculovirus caspase-inhibitory protein p35. A comparative two-dimensional gel proteomic analysis of empty vector- and p35-transfected cells after 12 h of exposure to 20 nm TPA, followed by mass spectrometry analysis, identified 38 differentially expressed proteins. Those overexpressed in p35-expressing cells (n = 16) were all full-length, whereas half of those overexpressed in control cells (n = 22) were N- or C-terminal cleavage fragments. The cleavage or degradation of seven of these proteins was confirmed in peripheral blood monocytes undergoing macrophage colony-stimulating factor-induced macrophagic differentiation. In U937 cells exposed to TPA, these proteolytic events can be inhibited by expression of a caspase-8 dominant negative mutant or the cowpox virus CrmA caspase inhibitor. These cleavages provide new insights to analyze the role of caspases in this specific differentiation program.

Induction of apoptosis and inhibition of telomerase activity by trichostatin A, a histone deacetylase inhibitor, in human leukemic U937 cells

The objective of the present study was to investigate the effect of trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, on the cell growth and apoptosis and its effect on the telomerase activity in human leukemic cell line U937. Exposure of U937 cells to TSA resulted in growth inhibition and induction of apoptosis in a dose-dependent manner as measured by hemocytometer counts, fluorescence microscopy, agarose gel electrophoresis and flow cytometry analysis. The increase in apoptosis was associated with the up-regulation in proapoptotic Bax expression and down-regulation of antiapoptotic Bcl-2 and Bcl-X L. TSA treatment inhibited the levels of cIAP family members and induced the proteolytic activation of caspase-3, which was associated with concomitant degradation of poly(ADP-ribose)-polymerase and β-catenin protein. TSA treatment markedly inhibited the activity of telomerase in a dose-dependent fashion. Additionally, the expression of human telomerase reverse transcriptase (hTERT), a main determinant of the telomerase enzymatic activity, was progressively down-regulated by TSA treatment. We therefore conclude that TSA demonstrated antiproliferative and apoptosis-inducing effects on U937 cells in vitro, and that changes in Bcl-2 family protein levels as well as telomerase activity may play an important role in its mechanism of action.

Cytotoxic and apoptogenic effect of tea ( Camellia sinensis var. assamica) root extract (TRE) and two of its steroidal saponins TS1 and TS2 on human leukemic cell lines K562 and U937 and on cells of CML and ALL patients

The anticancer activity of di- and tri-terpenes and other polyphenolic compounds present in tea is already reported. We evaluated the cytotoxic and apoptogenic effect of tea root extract (TRE) and two of its steroidal saponins named as TS1 and TS2, on human cell lines and on cells from leukemic patients. It was found that TRE, TS1 and TS2 significantly decreased cell count and that TRE caused apoptosis, as confirmed morphologically by confocal microscopy and by flow-cytometric analysis using Annexin-V FITC and propidium iodide (PI). Cell count and MTT assay in normal white blood cells (WBC) of healthy volunteers revealed that TRE produced insignificant reduction in cell count and cytotoxicity.