Hydroxydibenzoylmethane induces apoptosis through repressing ornithine decarboxylase in human promyelocytic leukemia HL-60 cells

Ornithine decarboxylase (ODC) is the rate-limiting enzyme in polyamine biosynthesis and a target for chemoprevention. Hydroxydibenzoylmethane (HDB), a derivative of dibenzoylmethane of licorice, is a promising chemopreventive agent. In this paper, we investigated whether HDB would inhibit the ODC pathway to enhance apoptosis in human promyelocytic leukemia HL-60 cells. We found ODC enzyme activity was reduced during HDB treatment. Overexpression of ODC in HL-60 parental cells could reduce HDB-induced apoptosis, which leads to loss of mitochondrial membrane potential (Δψ(m)), through lessening intracellular ROS. Furthermore, ODC overexpression protected cytochrome c release and the activation of caspase-3 following HDB treatment. The results demonstrated HDB-induced apoptosis was through a mechanism of down-regulation of ODC and occurred along a ROS-dependent mitochondria-mediated pathway.     

Induction of apoptosis in human promyelocytic leukemia HL60 cells by panaxynol and panaxydol

Panaxynol and panaxydol are naturally occurring polyacetylenes, isolated from the lipophilic fractions of Panax notoginseng, that exert anti-proliferative effects against malignant cells. However, to the best of our knowledge, no study concerning the inhibitory effects of the two polyacetylenes on cell growth of human promyelocytic leukemia cells has been reported. In this paper, we examined the antiproliferation and proapoptotic effects of panaxynol and panaxydol on HL60 cells and investigated their mechanism of action. Cell growth inhibition of panaxynol and panaxydol were determined by trypan blue dye exclusion assays. Apoptosis of cells was revealed by morphological observation, analysis for nuclear DNA distribution and by annexin V-FITC/ PI staining using flow cytometry. It was found that panaxynol and panaxydol markedly inhibited proliferation of HL60 cells in a time- and dose-dependent manner via an apoptotic pathway. In concern with these ?ndings, Western blot analysis showed proteolytic activation of PKCδ, caspase-3 activation and cleavage of poly (ADP [adenosine diphosphate]-ribose) polymerase in HL60 cells treated by panaxynol and panaxydol. In conclusion, panaxynol and panaxydol have profound effects on growth and apoptosis of HL60 cells, suggesting those substances are worthy of further exploration as potential anti-cancer agents.     

Regulation of bcl-2 family in hydrogen peroxide-induced apoptosis in human leukemia HL-60 cells

Numerous types of cells have been shown to undergo apoptosis when exposed to oxidant agent such as hydrogen peroxide. In order to understand the functional relationship between the anti- and pro-apoptotic regulatory proteins in the cells under oxidant stress, we have studied the level of expression of apoptosis regulatory proteins, bcl-2 and bax, in human leukemia HL-60 cells. The exposure of HL-60 cells to different concentrations of H2O2 for 6 h resulted in a typical apoptosis of the cells as characterized by flow cytometry, cell cycle analysis, and DNA fragmantation. There was a block in G1 to S transition and apoptotic cells were mainly derived from S and G2 cells. Kinetic study demonstrated that the levels of both bcl-2-mRNA and -protein expression were decreased with the progression of cellular apoptosis whereas the level of bax-mRNA was unchanged but the expressed bax-protein was not detectable. Cycloheximide, a nonspecific translation inhibitor, did not prevent the hydrogen peroxide-mediated apoptosis in HL-60 cells. These results suggest that the regulation of bcl-2, but not of bax are important factor in the oxidative stress-induced apoptosis in HL-60 cells.     

Resveratrol derivatives potently induce apoptosis in human promyelocytic leukemia cells

Resveratrol has been shown to possess antioxidant and anticancer activities, but little is known on the effect of resveratrol derivatives. Recently we have isolated resveratrol and its dimers and trimers from peony (Paeonia lactiflora) seeds, and reported their strong antioxidant and cytotoxic activity. In the present study, we have evaluated cellular effects of resveratrol derivatives; viniferin, gnetin H, and suffruticosol B on the proliferation and apoptosis in HL-60 cells in vitro. All resveratrol and its derivatives reduced viability of HL-60 cells in a dose-dependent manner with their IC(50) values of 20-90 microM. Ascending orders of IC(50) values were suffruticosol B, gnetin H, viniferin and resveratrol respectively. HL-60 cells treated with the four stilbenes exhibited the distinct morphological changes characteristics of cell apoptosis such as chromatin condensation, apoptotic bodies, and DNA fragmentations. A time-dependent histogram of the cellular DNA analyzed by flow cytometry revealed a rapid increase in subdiploid cells and a concomitant decrease in diploid cells exposed to 100 microM resveratrol for 0-24 h. Cells treated with 25 microM of resveratrol, viniferin, gnetin H, and suffruticosol B for 24 h resulted in increment of sub-G1 population by 51, 5, 11 and 59%, respectively. Treatment of cells with 0-20 microM resveratrol for 5 h produced a concentration-dependent decrease in cytochrome P450 (CYP) 1B1 mRNA levels. Suffruticosol B also suppressed CYP1B1 gene expression. These results demonstrated that resveratrol oligomers also strongly suppressed HL-60 cell proliferation, and induced DNA damage. In addition, CYP1B1 gene supression may suggest an involvement in the resveratrol-induced apoptosis in HL-60 cells.     

Effect of BCL-2 on Harringtonine-induced apoptosis and intracellular Ca2+ mobilization in human leukemia HL-60 cells

Harringtonine (HT), a kind of anticancer drug isolated from Chinese herb Cephalotaxus hainanensis Li, has been used in the clinical treatment of human glanulocytic leukemia and chromic myelocytic leukemia. In this study, we investigated the effect of Bcl-2 on HT-induced apoptosis and Ca²⁺ mobilization in human leukemia HL-60 cells. 1 g/ml HT induced the apoptosis of HL-60/Neo cells in a time-dependent manner; while 1 g/ml HT failed to induce the apoptosis of HL-60/ Bcl-2 cells. HT-, A23187- (a Ca²⁺ ionophore), Carbonyl cyanide m-chlorophenylhydrazone (CCCP,a specific releaser of Ca²⁺ from mitochondria) and thapsigargin- (an inhibitor of endoplasmic reticulum Ca^2+> -ATPase) induced changes in [Ca²⁺]_i were monitored by using Fluo 3-AM with confocal laser scanning microscopy. The results demonstrated that HL-60 cells with enforced expression of Bcl-2 (HL-60/Bcl-2 cells) had increased Ca²⁺ permeability and increased intracellular Ca²⁺ store in comparison with HL-60 cells with negative control vectors (HL-60/Neo cells), suggesting that Bcl-2 might prevent HT-induced apoptosis by increased Ca²⁺ permeability and increased intracellular Ca²⁺ buffering capacity.     

Induction of differentiation of human leukemia cells by various combinations of cytokines and low-molecular-weight inducers

To explore agents for differentiation therapy of leukemias, various combinations of cytokines and low-molecular-weight inducers were examined for differentiation-inducing activity toward three kinds of human leukemia-derived cell lines. The strongest differentiation inducing activity on promyelocytic HL60 cells and histiocytic U937 cells was obtained by combining recombinant tumor necrosis factor (rTNF), interferon-gamma (IFN-gamma), retinoic acid (RA), and 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3). For myeloblastic ML1 cells, the combination of rTNF, IFN-gamma, and RA had the strongest differentiation-inducing activity.     

Proteomic analysis of apoptosis initiation induced by all-trans retinoic acid in human acute promyelocytic leukemia cells

The irreversible destiny of apoptosis in its early stage might play a critical role in the apoptosis of human acute promyelocytic leukemia (APL) cell line induced by all-trans retinoic acid (ATRA). To characterize protein alterations during the apoptosis-initiation phase and to understand the metabolic status at that time, we investigated the protein profiles in the apoptosis-initiation phase of APL cell line HL-60 by proteomic analysis. ATRA-withdrawal was conducted to demonstrate that there was committed initiation phase of apoptosis triggered by 10(-6) M ATRA at day 3. Only after that time point, ATRA-treated cells irreversibly went to apoptosis. Also at that time point, the positive regulators of apoptosis such as STAT3 increased at protein level, whereas negative regulators (Bcl-2 and p-STAT3) decreased. In addition, caspase-3 also increased after that time. Furthermore, comparative proteomic analysis was utilized to examine the protein expression profiles during the initiation stage of apoptosis. Our results showed 12 upregulated and 7 downregulated proteins experiencing twofold alteration, including key regulators of signal transduction such as G-proteins and nucleic receptors, proteins related with metabolism, oxidation and reduction, proteins associated with the nucleus and cytoskeleton-related proteins. Some of them could be positive modulators to trigger apoptosis, whereas others could contribute to intracellular defense against apoptosis induced by exogenous triggers. The results above suggest that there is a subtle balance between apoptosis and the intracellular defense against apoptosis. Once the balance is disturbed, cells would irreversibly initiate to undergo the execution of apoptosis.     

Acidity of hydroxamic acids and amides

The relatively strong acidity of hydroxamic acids was analyzed by means of isodesmic reactions in which this acid or its anion is formed from simpler precursors. Acidity of amides was analyzed in the same way. Energies of all compounds involved in the reactions were calculated at the B3LYP/AUG-cc-pVTZ//B3LYP/6-311 + G(d,p) level; at this level a good agreement was reached with the sparse experimental data. Interpretation of the results was the same as in the recent discussion of the acidity of carboxylic acids, and the conclusions were similar: both amides and hydroxamic acids are stabilized with respect to simpler reference molecules of amines or N-alkylhydroxylamines, respectively. However, their anions are stabilized still more and are responsible for the acidity. This effect is stronger in hydroxamic acids or amides than in carboxylic acids. The problem of whether it is due to resonance depends on the definition of this term. Semiquantitative comparison suggests that resonance in hydroxamic acids is more important than in amides and still more than in carboxylic acids. The stronger acidity of hydroxamic acids compared to amides is due to the destabilizing inductive effect of the hydroxyl group in the acid molecule, not to any effect in the anion.     

A new daphnane diterpenoid from Excoecaria venenata with inhibitory effect on human leukaemia HL-60 cells

A new daphnane diterpenoid, venenatin (1), along with six known compounds, was isolated from the aerial parts of Excoecaria venenata. The structure of 1 was elucidated on the basis of extensive spectroscopic analysis. Compound 1 exhibited growth inhibitory effect on human leukaemia HL-60 cell line with IC₅₀ value of 28.10 μM.     

Expression of hOGG1 protein during differentiation of HL-60 cells

Human 8-oxo-G-DNA glycosylase 1 (hOGG1) is a DNA glycosylase to cleave 8-oxo-7,8-dihydroguanine (8-oxo-G), a mutagenic DNA adduct formed by oxidant stresses. Here, we examined hOGG1 protein expression and repair activity to nick a DNA strand at the site of 8-oxo-G during differentiation of hematopoietic cells using HL-60 cells. Overall expression of hOGG1 protein was increased during granulocytic differentiation of HL-60 cells induced by DMSO and monocytic differentiation by vitamine D(3). Greater level of hOGG1 protein was expressed in DMSO-treated cells. However, change in the DNA nicking activity was not in parallel with the change in hOGG1 protein expression, especially in PMA-treated cells. In PMA- treated cells, the level of hOGG1 protein was lowered, even though the DNA nicking activity was elevated, in a manner similar to the changes in serum- deprived HL-60 cells. These results indicate that hOGG1 expression change during differentiation of hematopoietic stem cells for adaptation to new environments. And the DNA cleaving activity may require additional factor(s) other than expressed hOGG1 protein, especially in apoptotic cell death.     

Apoptosis and membrane permeabilisation induced by macranthoside B on HL-60 cells

Triterpene saponins are throught to be potential anti-tumour agents in many cell types. This study aims to evaluate the cytotoxic activity and mechanism of a triterpene saponin, macranthoside B (MB), isolated from Lonicera macranthoides Hand.-Mazz. (Caprifoliaceae). A cell viability assay showed that MB inhibited cell growth of a panel of six cancer cell lines, especially in human acute promyelocytic leukaemia HL-60 cells, with an IC50 value of 3.8 μmol. A hypodiploid cells assay and an annexin-V-FITC/PI double staining assay showed a significant increase of apoptosis in a dose-dependent manner on HL-60 cells both 24 and 48 h after MB treatment. MB-induced apoptosis was through the caspase-mediated pathway, by activation of caspase-3. Furthermore, a lactate dehydrogenase (LDH) release test suggested that an MB-cholesterol interaction led to the rearrangement of the lipid bilayer and to subsequent cell membrane impairment. Taken together, these findings demonstrate that MB may exhibit cytotoxic activity against HL-60 cells by inducing apoptosis via caspase-dependent pathways and also membrane permeabilisation.     

Change in electrophoretic mobility of HL-60RG cells by apoptosis

We measured the electrophoretic mobilities of HL-60RG cells and their apoptotic cells triggered by Actinomycin D as a function of the ionic strength of the suspending medium at pH 7.4. Both types of cells showed negative mobilities. The apoptotic HL-60RG cells exhibited larger mobility values in magnitude than intact HL-60RG cells in the whole range of the electrolyte concentration measured. The obtained data were analyzed via a mobility expression for soft particles, that is, colloidal particles with ionpenetrable surface layers. The observed mobility difference between the intact and apoptotic HL-60RG cells was found to be due mainly to the difference in friction exerted by the cell surface layers on the liquid flow around the cells between these two types of cells rather than the difference in charge density in their surface layers. A possible explanation for this mobility change by apoptosis is given.     

2beta-(N-substituted piperazino)-5alpha-androstane-3alpha,17beta-diols: parallel solid-phase synthesis and antiproliferative activity on human leukemia HL-60 cells

Leukemia is the most common cancer affecting children. A steroid possessing a methylpiperazine nucleus was recently reported to inhibit the proliferation of HL-60 leukemia cells. To speed up the development of this promising potential new drug, we generated libraries of analogues using parallel solid-phase organic synthesis (SPOS). A 6-step sequence of reactions, starting from dihydrotestosterone, afforded a steroidal 2,3alpha-epoxide, which was selectively opened to give, after N-Fmoc protection, a diol with suitable stereochemistry. The difference of reactivity between 3alpha-OH and 17beta-OH was then used to allow the regioselective coupling of 17beta-OH to chloro-activated butyldiethylsilane polystyrene. We next generated three libraries of 2beta-piperazinyl-5alpha-androstane-3alpha,17beta-diol N-derivatives with 1, 2, or 3 levels of molecular diversity in acceptable yields and purities for our biological screening assay. Several members of these libraries were more potent than the lead compound, especially five members with a proline as the first level of diversity and a cyclohexylcarbonyl, methylbutyryl, cyclohexylacetyl, cyclopentylpropionyl, or hexanoyl as the second level of diversity. They efficiently inhibited HL-60 cell proliferation with IC50 values of 0.58, 0.66, 1.78, 1.98, and 2.57 microM, respectively. The present work demonstrates the potential of our SPOS approach for the optimization of a new class of cytotoxic agents.     

Effects of static magnetic field on human leukemic cell line HL-60

A number of structures with magnetic moments exists in living organisms that may be oriented by magnetic field. While most experimental efforts belong to the area of effects induced by weak and extremely low-frequency electromagnetic fields, we attempt to give an attention to the biological effects of strong static magnetic fields. The influence of static magnetic field (SMF) on metabolic activity of cells was examined. The metabolic activity retardation is observed in human leukemic cell line HL-60 exposed to 1-T SMF for 72 h. The retardation effect was observed as well as in the presence of the mixture of the antineoplastic drugs 5 fluorouracil, cisplatin, doxorubicin and vincristine.     

Unnatural amino acids. 3. Aziridinyl ketones from esters and amides of aziridine-2-carboxylic acids

A series of N-substituted amides and esters of aziridine-2-carboxylic acids have been prepared and have been subjected to deprotonation with lithium diisopropylamide. The intermediate carbanions reacted more readily with the carbonyl groups of the substrates than with methyl iodide. So, in place of the expected amides or esters of methylaziridine-2-carboxylic acids, amides or esters of 2-aziridinylcarbonylaziridine-2-carboxylic acids were isolated. __________ Translated from Khimiya Geterotsiklicheskikh Soedinenii, No. 2, 220–225, February, 2007.