Viability Of HL-60 Cells Research Articles

Supercritical carbon dioxide extraction enhances flavonoids in water-soluble propolis

Two propolis, obtained from Brazil and China, were ground and individually treated using supercritical carbon dioxide to enhance the extraction and water solubility of nine flavonoids. Co-solvent effects of ethanol and water were examined. Extractions were carried out semi-batchly at 5 mL/min CO2, at pressures ranging from 2000 to 5000 psig, at temperatures ranging from 35 to 65℃, and with propolis to co-solvent ratios of 1:2 and 1:5 (w/v). After supercritical extraction of 500 liters of CO2, the residual propolis in the extractor was further extracted by means of normal solvent extraction using 95% ethanol (ReE extract was obtained) and de-ionized water (ReW extract was obtained). Nine active propolis flavonoids in the extracts were analyzed to verify their inhibitory effects on leukemia cancer cells. Experimental results indicated that the dry SC-CO2 extract mainly contained low-polar wax-like materials. The crude yield of Brazilian propolis in the dry SC-CO2 extract reached around 10% at 45℃ and 4000 psig extraction. The amounts of the nine flavonoids in the Brazilian ReE and ReW extracts were 5% and 120%, higher than those obtained by means of conventional ethanol and water extraction, respectively. The Brazilian ReW extracts clearly contained a few water-soluble compounds and, especially, enhanced amounts of the nine flavonoids. All the extracts were tested for viability and cell proliferation of leukemia HL-60 and U937 cancer cells, and most exhibited a postivie inhibitory effect. The results also suggested that something other than the nine flavonoids in dry SC-CO2 extract was importantly involved in the inhibitory effect.

Sodium salicylate-triggered apoptosis in HL-60 cells depends on caspase-8 activation.

For investigation of the killing and proapoptotic effects of sodium salicylate (Na-Sal) on HL-60 cells, the cytotoxic activity of Na-Sal was measured by means of MTT assay. Apoptosis was identified and analyzed with the help of transmission electron microscopy, annexin V staining, and DNA gel electrophoresis, and the association of caspase-8 activation with apoptosis was determined with the specific protease inhibitor IETD-fmk. After exposure of HL-60 cells to increasing concentrations of Na-Sal (0.5, 1, 3, 5, and 7 mmol/L) for 24 hours, the mean cell viability gradually dropped to 92%, 83%, 68%, 50%, and 42%. With treatment of target cells with 5-mmol/L (IC50) Na-Sal for 6, 12, 24, or 36 hours, the mean cell survival tapered to 91%, 81%, 48% (P <.05 versus control), and 14% (P <.05 versus control). Again incubated with 5-mmol/L Na-Sal for 12 or 24 hours, HL-60 cells displayed clear early or late signs of apoptosis, including (1) notable enhancement of phosphatidylserine externalization, (2) cell shrinkage, membrane blebbing, and eventual disintegration into numerous apoptotic bodies, and (3) formation of ladder DNA. The viability of HL-60 cells increased significantly during 24 or 36 hours of coculture with 100-micromol/L IETD-fmk in combination with 5-mmol/L Na-Sal compared with the viability when 5-mmol/L Na-Sal was used alone (P < .05). Moreover, the target cells showed a considerable decrease in phosphatidylserine exposure and DNA fragmentation after coincubation for 12 or 24 hours performed as described above. The findings presented herein strongly suggest that Na-Sal can exert potent killing and proapoptotic activity against HL-60 cells, and this effect appears to depend on caspase-8 activation.

A novel plasma membrane quinone reductase and NAD(P)H:quinone oxidoreductase 1 are upregulated by serum withdrawal in human promyelocytic HL-60 cells.

We have studied changes in plasma membrane NAD(P)H:quinone oxidoreductases of HL-60 cells under serum withdrawal conditions, as a model to analyze cell responses to oxidative stress. Highly enriched plasma membrane fractions were obtained from cell homogenates. A major part of NADH-quinone oxidoreductase in the plasma membrane was insensitive to micromolar concentrations of dicumarol, a specific inhibitor of the NAD(P)H:quinone oxidoreductase 1 (NQOI, DT-diaphorase), and only a minor portion was characterized as DT-diaphorase. An enzyme with properties of a cytochrome b5 reductase accounted for most dicumarol-resistant quinone reductase activity in HL-60 plasma membranes. The enzyme used mainly NADH as donor, it reduced coenzyme Q0 through a one-electron mechanism with generation of superoxide, and its inhibition profile by p-hydroxymercuribenzoate was similar to that of authentic cytochrome b5 reductase. Both NQO1 and a novel dicumarol-insensitive quinone reductase that was not accounted by a cytochrome b5 reductase were significantly increased in plasma membranes after serum deprivation, showing a peak at 32 h of treatment. The reductase was specific for NADH, did not generate superoxide during quinone reduction, and was significantly resistant to p-hydroxymercuribenzoate. The function of this novel quinone reductase remains to be elucidated whereas dicumarol inhibition of NQO1 strongly potentiated growth arrest and decreased viability of HL-60 cells in the absence of serum. Our results demonstrate that upregulation of two-electron quinone reductases at the plasma membrane is a mechanism evoked by cells for defense against oxidative stress caused by serum withdrawal.

In vitro testing of the potential for orthopedic bone cements to cause apoptosis of osteoblast-like cells

The purpose of this study was to investigate in vitro the apoptosis- and/or necrosis-inducing potential of polymethylmethacrylate (PMMA)-based bone cements for prosthetic surgery. Four bone cements widely used in orthopedics were tested as extracts onto osteoblast-like MG-63 cells and for comparison, HL-60 cells, which are remarkably sensitive to apoptotic stimuli. Neutral red uptake (NRU) was used to measure cell viability, while Hoechst 33258 staining was used to detect DNA content. Apoptosis was characterized using a BrdU-based ELISA assay for DNA fragmentation and examined by fluorescence microscopy using acridine orange and propidium iodide staining of nuclei. The generation of reactive oxygen species (ROS), which could mediate apoptosis, was verified using dichlorofluorescein-diacetate (DCFH-DA) oxidation to DCF. After 24 h of challenge of the cells with the four cement extracts, the viability of either MG-63 or HL-60 cells was found to be unaltered, as recorded by NRU. Apoptotic cell death was induced by three cements in HL-60, whereas MG-63 cells were significantly affected by the four cements tested: the finding of DNA fragments both in the cytoplasm and supernatants of MG-63 after 24 h demonstrated that these cells underwent late-apoptosis/secondary necrosis. Fluorescent staining of the nuclei confirmed the results obtained with the ELISA test. Oxygen free radicals were elicited by two cements in HL-60 cells, while MG-63 did not generate ROS in response to cements. This study helps to gain more insight into the mechanism of cell death induced by PMMA-based cements and suggests apoptosis of osteoblasts as a part of the tissue reaction around cemented prostheses.

Eupatilin, a pharmacologically active flavone derived from Artemisia plants, induces apoptosis in human promyelocytic leukemia cells

Extracts of the whole herb of Artemisia asiatica Nakai (Asteraceae) have been used in traditional oriental medicine for the treatment of inflammation, cancer and other disorders. In the present work, we have evaluated the apoptosis-inducing capability of eupatilin (5,7-dihydroxy-3,4,6-trimethoxyflavone), a pharmacologically active ingredient of A. asiatica, in cultured human promyelocytic leukemia (HL-60) cells. Thus, eupatilin exhibited concentration-dependent inhibitory effects on viability and DNA synthesis capability of HL-60 cells. The anti-proliferative effect of eupatilin was attributable to its apoptosis-inducing activity as determined by characteristic nuclear condensation, in situ terminal end-labeling of fragmented DNA (TUNEL), release of mitochondrial cytochrome c into cytoplasm, proteolytic activation of caspases-9, -3, and -7, and cleavage of poly(ADP-ribose)polymerase. Eupatilin-induced HL-60 cell apoptosis does not appear to be mediated via alteration in Bcl-2/Bax-2. Taken together, the above findings suggest that eupatilin has chemopreventive and cytotoxic effects.

Quinolizin-Coumarins as Physical Enhancers of Chemiluminescence during Lipid Peroxidation in Live HL-60 Cells

We investigated whether physical enhancers of low-level chemiluminescence—coumarin laser dyes C-314, C-334, and C-525—may be used to monitor interactions of lipid peroxyl radicals during lipid peroxidation in live cells. We present data demonstrating that two quinolizin-substituted coumarins—C-525 and C-334—can be integrated into HL-60 cells and successfully used as physical enhancers of chemiluminescence induced by the lipid soluble azo-initiator 2,2′-azobis(2,4-dimethyl-valeronitrile) (AMVN). Coumarins did not inhibit AMVN-induced peroxidation of membrane phospholipids in HL-60 cells, and no consumption of these coumarins occurred in the course of AMVN-induced oxidative stress. Redox status, evaluated by intracellular GSH content, remained unchanged after treatment with the coumarins. tert-Butyl hydroperoxide and cumene hydroperoxide (more hydrophilic oxidants) induced a lower chemiluminescence signal with both coumarins. Viability of HL-60 cells was not affected by coumarins both in the presence and in the absence of oxidants. Based on these results we conclude that quinolizin-substituted coumarins represent a promising class of physical enhancers of chemiluminescence for monitoring free radical peroxidation in live cells.

Liposomal Bcl-2 antisense oligonucleotides enhance proliferation, sensitize acute myeloid leukemia to cytosine-arabinoside, and induce apoptosis independent of other antiapoptotic proteins

AbstractThe antiapoptotic proteins, Bcl-2 and Bcl-XL, are expressed in most cases of acute myeloid leukemia (AML) and may contribute to drug resistance in AML. We tested the hypothesis that down-regulation of Bcl-2 alone by antisense oligodeoxynucleotides (Bcl-2-AS) induces apoptosis, even in the presence of other antiapoptotic genes. We tested Bcl-2-AS in myeloid leukemic HL-60 cells, in Bcl-2 and Bcl-XL overexpressing HL-60-DOX cells, and in primary AML samples. Down-regulation of Bcl-2 by Bcl-2-AS reduced the viability of HL-60 cells and, less effectively, HL-60-DOX cells and increased ara-C cytotoxicity in both cell lines. Incubation of primary AML blasts with Bcl-2-AS decreased Bcl-2 expression in CD34+ blast cells after induction of apoptosis and enhancement of ara-C cytotoxicity in 11 of 19 primary AML samples. In 8 samples in which Bcl-2-AS did not induce apoptosis, baseline Bcl-2 levels were found to be strikingly high. The expression of other antiapoptotic proteins (Bcl-XL, Bag-1, A1, and Mcl-1) did not prevent Bcl-2-AS–induced apoptosis. Bcl-2-AS also inhibited colony formation of AML progenitor cells. Low concentrations of Bcl-2-AS induced significant increases in S-phase cells (P = .04). Results establish Bcl-2 as a critical target for AS strategies in AML in which the baseline levels predict response to Bcl-2-AS. Bcl-2 exerts both antiapoptotic and antiproliferative functions in AML. Because early normal hematopoietic stem cells do not express Bcl-2, Bcl-2-AS therapy should be highly selective for AML cells.

Liposomal Bcl-2 antisense oligonucleotides enhance proliferation, sensitize acute myeloid leukemia to cytosine-arabinoside, and induce apoptosis independent of other antiapoptotic proteins

The antiapoptotic proteins, Bcl-2 and Bcl-XL, are expressed in most cases of acute myeloid leukemia (AML) and may contribute to drug resistance in AML. We tested the hypothesis that down-regulation of Bcl-2 alone by antisense oligodeoxynucleotides (Bcl-2-AS) induces apoptosis, even in the presence of other antiapoptotic genes. We tested Bcl-2-AS in myeloid leukemic HL-60 cells, in Bcl-2 and Bcl-XL overexpressing HL-60-DOX cells, and in primary AML samples. Down-regulation of Bcl-2 by Bcl-2-AS reduced the viability of HL-60 cells and, less effectively, HL-60-DOX cells and increased ara-C cytotoxicity in both cell lines. Incubation of primary AML blasts with Bcl-2-AS decreased Bcl-2 expression in CD34+ blast cells after induction of apoptosis and enhancement of ara-C cytotoxicity in 11 of 19 primary AML samples. In 8 samples in which Bcl-2-AS did not induce apoptosis, baseline Bcl-2 levels were found to be strikingly high. The expression of other antiapoptotic proteins (Bcl-XL, Bag-1, A1, and Mcl-1) did not prevent Bcl-2-AS–induced apoptosis. Bcl-2-AS also inhibited colony formation of AML progenitor cells. Low concentrations of Bcl-2-AS induced significant increases in S-phase cells (P = .04). Results establish Bcl-2 as a critical target for AS strategies in AML in which the baseline levels predict response to Bcl-2-AS. Bcl-2 exerts both antiapoptotic and antiproliferative functions in AML. Because early normal hematopoietic stem cells do not express Bcl-2, Bcl-2-AS therapy should be highly selective for AML cells.

Induction of apoptosis by idarubicin: how important is the plasma peak?

It is still not clear whether a high plasma peak or a prolonged plasma presence of the drug is optimal for chemotherapy with anthracyclines. A high plasma peak seems to correlate with the liberation of oxygen radicals and cumulative delayed cardiotoxicity, and therefore, should be avoided. On the other hand, its role in attaining the desired therapeutic effect, i.e. induction of apoptosis in tumor cells, has not been clearly elucidated. Idarubicin is the only anthracycline that can be applied orally. We measured the DNA-binding of idarubicin and idarubicinol and the induced apoptosis in the human promyelocytic HL-60 leukemia cell line. Various pharmacokinetic profiles, with and without clinically relevant peak concentrations, were simulated in vitro. The concentration necessary for maximal DNA-binding and subsequent induction of apoptosis was 1.5 microg/ml for 20 minutes which is well above the plasma concentration achievable in therapy. A plateau of apoptosis was observed after 90 minutes of incubation; a prolongation above 90 minutes did not increase the rate of apoptosis. We simulated a bolus application and a continuous infusion using two different pharmacokinetic profiles of idarubicin with comparable AUCs (area under the time curve). After 48 hours of total incubation, the viability of HL-60 cells was 56.88% with profile 1 (50 ng/ml idarubicin for 2 hours) and 83.00% with profile 2 (4.25 ng/ml for 24 hours). Although these in vitro experiments are not directly applicable to the clinical situation, they do indicate that a prolongation of the application time up to at least 90 minutes, either by continuous infusion or by oral application, may be acceptable as a method of increasing apoptosis. On the other hand, the plasma peak seems to be an important factor for the induction of apoptosis. Further studies are in progress to define the minimal plasma peak necessary to induce a maximum of apoptosis.

Protective Effects of Glucocorticoids on Taxol-Induced Cytotoxicity in Human Leukemia Hl-60 Cells

Although the therapeutic actions of glucocorticoids are largely attributed to anti-inflammatory and immunosuppressive effects, they have been implicated in enhancing tissue and cellular protections. In this study, we examined whether glucocorticoids including dexamethasone (Dex) and hydroxy-cortisone could diminish the cytotoxic effects of anti-microtubule agents including taxol (paclitaxel), microtubule stabilizing agent, and colchicine, microtubule disrupting agent, on human leukemia HL-60 cells. Taxol or colchicine decreased the viability of HL-60 cells in a dose-dependent manner. However, micromolar concentrations of glucocorticoids rendered HL-60 cells resistant against the cytotoxic activity of anti-microtubule agents. Pretreatments of the glucocorticoids were more effective than simultaneous treatments with antimicrotuble agents. The fact that actinomycin D or cycloheximide reversed the cytoprotective effects of glucocorticoids on cytotoxicities in HL-60 cells induced by antirnicrotuble agents suggests glucocorticoids cytoprotection might be mediated via newly synthesized protein. Collectively, these data showed that micromolar concentrations of dexamethasone or hydrocortisone could attenuate the cytotoxic effects of taxol or colchicine on human leukemia HL-60 cells via protein synthesis.

The effect of pyrimidine nucleosides on adenosine-induced apoptosis in HL-60 cells.

Among several nucleosides, adenosine is the only one to induce typical apoptotic cell death in human promyelocytic leukemia HL-60 cells. Intracellularly transported adenosine seemed to be required for the induction of apoptosis, since dipyridamole, which inhibits the transport of adenosine, strongly suppressed apoptosis, and 8-phenyltheophylline, a receptor antagonist, did not affect the adenosine-induced effect. The viability of adenosine-treated HL-60 cells was partially recovered by supplementation with a pyrimidine nucleoside, uridine or thymidine. Cytidine or deoxycytidine had no effect on the growth and survival of adenosine-treated cells, while uridine or thymidine inhibited adenosine-induced intracellular DNA fragmentation. These results suggest that the quantitative adjustment of purine and pyrimidine nucleosides might play an important role in the adenosine-induced apoptosis of HL-60 cells. The reduction of c-Myc expression in adenosine-treated cells was prevented by uridine or thymidine. These observations suggest that the expression of c-Myc might be related to an intracellular sensing system for the quantitative balance of nucleosides or nucleotides.

Cytotoxicity of solid lipid nanoparticles as a function of the lipid matrix and the surfactant.

Assessment of the in vitro cytotoxicity of solid lipid nanoparticles (SLNs) as a function of lipid matrix (Dynasan 114, Compritol ATO 888), and stabilizing surfactant (poloxamers, Tween 80, soya lecithin, and sodium dodecyl sulphate). Comparison with other colloidal carriers should determine their potential use in the clinic. SLNs were produced by high pressure homogenisation. Cytotoxicity was assessed by measuring the viability of HL60 cells and human granulocytes after incubation with SLNs. Particle internalisation was quantified by chemiluminescence measurements. The nature of the lipid had no effect on viability; distinct differences were found for the surfactants. Binding to the SLN surface reduced markedly the cytotoxic effect of the surfactants, e.g., up to a factor of 65 for poloxamer 184. The permanent HL60 cell line-differentiated from cells with granulocyte characteristics by retinoic acid treatment-yielded results identical to freshly isolated human granulocytes. In general, the SLNs showed a lower cytotoxicity compared to polyalkylcyanoacrylate and polylactic/glycolic acid (PLA/ GA) nanoparticles. Because the results are identical when using human granulocytes, differentiated HL60 cells can be used as an easily accessible in vitro test system for i.v. injectable SLN formulations. The SLNs appear suitable as a drug carrier system for potential intravenous use due to their very low cytotoxicity in vitro.

Inhibition of leukemia cell proliferation by receptor-mediated uptake of c-myb antisense oligodeoxynucleotides.

Exposure of human leukemia HL-60 cells to an oligodeoxynucleotide complementary to an 18-base sequence (codons 2-7) of c-myb-encoded mRNA has previously been shown to result in inhibition of cell proliferation. Because HL-60 cells express high levels of transferrin receptor we adapted a DNA delivery system based on receptor-mediated endocytosis to introduce myb oligomers complexed with a transferrin-polylysine conjugate into those cells. A DNA.RNA duplex resistant to S1 nuclease digestion was detected as early as 12 hr after culture of HL-60 cells in the presence of the myb antisense/transferrin-polylysine complex. Exposure of HL-60 cells to the myb antisense/transferrin-polylysine complex resulted in rapid and profound inhibition of proliferation and loss of cell viability much more pronounced than that occurring in cells exposed to free myb antisense oligodeoxynucleotides. The transferrin-polylysine/myb sense complex or the transferrin-polylysine conjugate alone had no effect on HL-60 cell proliferation and viability. These findings indicate that myb synthetic oligodeoxynucleotides enter efficiently into HL-60 by transferrin receptor-mediated endocytosis and exert a profound biological effect. Such a delivery system could exploit other ligand-receptor interactions for the selective delivery of oncogene-targeted antisense oligodeoxynucleotides.

Ｌ‐９２９細胞のコロニー形成試験を用いた水中微量汚染物質の毒性評価

Colony formation assay using L-929 cells was applied to 40 chemicals which were reported to occur in drinking water. In the 40 chemicals including 4 of specified chemical substances, 7 of pesticides, 19 of industrial chemicals and 10 of halogenated hydrocarbons, some of pesticides and halogenated hydrocarbons inhibited strongly the colony formation of L-929 cells, and the number and position of halogen in the chemicals influenced their cytotoxicities. The results were compared with those by the other cytotoxicity tests involving viability of HL-60 cells, phagocytic activity of mouse peritoneal macrophages, and LDH release or glycogenolytic activity of rat hepatocytes, and cytotoxicity matrix were obtained from the comparison of these four cytotoxicity tests. Availability of the cytotoxicity matrix is discussed to estimate the micropollutants in water.