L-arginine and mitomycin C-induced nitric oxide release and apoptosis in human lymphocytes

Nitric oxide (NO) is a free radical that is produced by a number of mammalian cell types from L-arginine and a critical mediator that acts in many tissues to regulate a diverse range of physiological processes. The major metabolic end product for NO is nitrate (NO(3)) and nitrite (NO(2)), which are stable metabolites within tissue, plasma, and urine. Measurements of nitrate and nitrite values reveal alterations in NO production. Endogenously generated or exogenously applied NO causes DNA cleavage by endonuclease activation.We investigated the effect of L-arginine and mitomycin C (MMC) on cultured lymphocytes of healthy individuals. We observed chromosome breaks, apoptotic cells and increased NO levels after L-arginine and MMC addition. In conclusion, our results confirmed that NO may be the cause of apoptotic cell death in L-arginine added lymphocyte culture.     

Apoptosis induced by (di-isopropyloxyphoryl-Trp)2-Lys-OCH3 in K562 and HeLa cells

According to the method used in our laboratory, our group synthesized (DIPP-Trp)₂-Lys-OCH₃. It inhibited the proliferation of K562 and HeLa cells in a dose-and time-dependent manner with an IC₅₀ of 15.12 and 42.23 µM, respectively. (DIPP-Trp)₂-Lys-OCH₃ induced a dose-dependent increase of the G2/M cell population in K562 cells, and S cell population in HeLa cells; the sub-G0 population increased dramatically in both cell lines as seen by PI staining experiments using a FACS Calibur Flow cytometer (BeckmanCoulter, USA). Phosphatidylserine could significantly translocate to the surface of the membrane in (DIPP-Trp)₂-Lys-OCH₃-treated K562 and HeLa cells. The increase of an early apoptotic population was observed in a dose-dependent manner by both annexin-FITC and PI staining. It was concluded that (DIPP-Trp)₂-Lys-OCH₃ not only induced cells to enter into apoptosis, but also affected the progress of the cell cycle. It may have arrested the K562 and HeLa cells in the G₂/M, S phases, respectively. The apoptotic pathway was pulsed at this point, resulting in the treated cells entering into programmed cell death. (DIPP-Trp)₂-Lys-OCH₃ is a potential anticancer drug that intervenes in the signalling pathway.     

Endogenous nitric oxide activation protects against cigarette smoking induced apoptosis in endothelial cells

Cigarette-induced endothelial dysfunction could be an early mediator of atherosclerosis. In this study, we explored the mechanisms of cigarette smoke extract (CSE)-induced human aortic endothelial cells (HAEC) apoptosis. We found that 10-65% of HAECs underwent apoptotic changes when HAECs were exposed to 0.001-0.02 cigarette equivalent unit of CSE for 4 h. CSE activated the caspases-3 and 8, the p38 MAP kinase and stress activated protein kinase/c-Jun N-terminal protein kinase (SAPK/JNK). Specific inhibitors of p38 MAP or SAPK/JNK reduced CSE-induced caspase activation. We further showed that eNOS pre-activation by L-arginine reduced endothelial apoptosis from 65% to 5%; and eNOS inhibition by N-omega-nitro-L-arginine methyl ester accentuated CSE-induced endothelial apoptosis. We suggest that appropriate endogenous NO production may be an important protective mechanism against smoking-induced endothelial damage.     

Metal release in metallothioneins induced by nitric oxide: X-ray absorption spectroscopy study

Metallothioneins (MTs) are low molecular weight proteins that include metal ions in thiolate clusters. The capability of metallothioneins to bind different metals has suggested their use as biosensors for different elements. We study here the interaction of nitric oxide with rat liver MTs by using in situ X-ray absorption spectroscopy techniques. We univocally show that the presence of NO induces the release of Zn atoms from the MT structure to the solution. Zn ions transform in the presence of NO from a tetrahedral four-fold coordinated environment in the MT into a regular octahedral six-fold coordinated state, with interatomic distances compatible with those of Zn solvated in water.     

Inducible nitric oxide synthase mediates MG132 lethality in leukemic cells through mitochondrial depolarization

Proteasomes are highly expressed in rapidly growing neoplastic cells and essential for controlling the cell cycle process and mitochondrial homeostasis. Pharmacological inhibition of the proteasome shows a significant anticancer effect on hematopoietic malignancies that is usually associated with the generation of reactive oxygen species. In this study, we comprehensively investigated the role of endogenous oxidants in various cellular events of K562 leukemic cells in response to treatment with MG132, a proteasome inhibitor. MG132 at 1.4 µM potently triggered G₂/M arrest, mitochondrial depolarization, and apoptosis. By such treatment, the protein level of inducible nitric oxide synthase (iNOS) was doubled and cellular oxidants, including nitric oxide, superoxide, and their derivatives, were increasingly produced. In MG132-treated cells, the increase in iNOS-derived oxidants was responsible for mitochondrial depolarization and caspase-dependent apoptosis, but was insignificant in G₂/M arrest. The amount of iNOS was negatively correlated with that of manganese superoxide dismutase (MnSOD). Whereas iNOS activity was inhibited by aminoguanidine, cellular MnSOD levels as well as mitochondrial membrane potentials were upregulated, and consequentially G₂/M arrest and apoptosis were thoroughly reversed. It is suggested that cells rich in functional mitochondria possess improved proteasome activity, which antagonizes the cytotoxic and cytostatic effects of MG132. In contrast to iNOS, endothelial NOS-driven cGMP-dependent signaling promoted mitochondrial function and survival of MG132-stressed cells. In conclusion, the functional interplay of proteasomes and mitochondria is crucial for leukemic cell growth, wherein iNOS plays a key role.     

Growth hormone induces eNOS expression and nitric oxide release in a cultured human endothelial cell line

Growth hormone deficiency is linked to cardiovascular disease and particularly increased peripheral vascular resistance. Surprisingly, its role in endothelial nitric oxide (NO) synthetase (eNOS) regulation and NO release is basically unknown. We therefore studied the effects of different doses of somatotropin in cultures of a human endothelial cell line (EAhy926). We investigated expression and activity of eNOS, as well as other target genes known to be deregulated in cardiovascular disease including E-selectin and the lectin-like oxidized low density lipoprotein receptor. Treatment of cultured human endothelial cells with somatotropin resulted in significant (P<0.05) increases of eNOS gene and protein expression, as well as NO release, whereas production of intracellular reactive oxygen species was significantly reduced, at the highest somatotropin dose level. The enhanced eNOS gene/protein expression and enzyme activity correlate well. Our findings are suggestive for a novel role of growth hormone in endothelial biology, and particularly NO production.     

Gamma-linolenic acid induces apoptosis and lipid peroxidation in human chronic myelogenous leukemia K562 cells

Various polyunsaturated fatty acids, especially gamma-linolenic acid (GLA), inhibit the growth of a variety of tumor cells. Some evidence indicates that polyunsaturated fatty acid can kill cells by apoptosis. In the current study, we tested the apoptotic effect of GLA on human chronic myelogenous leukemia K562 cells. GLA induced K562 cell death in a dose-dependent manner. Typical apoptotic nuclei were shown by staining of K562 cells with DNA-binding fluorochrome Hoechst 33342, characterized by chromatin condensation and nuclear fragmentation. Flow cytometric analysis also demonstrated that GLA caused dose-dependent apoptosis of K562 cells. The apoptosis could be inhibited by a pancaspase inhibitor (z-VAD-fmk), suggesting the involvement of caspases. Further, release of cytochrome c, activation of caspase-3 and cleavage of PARP were found in GLA-induced apoptosis. GLA treatment could also elevate lipid peroxidation in K562 cells, and antioxidant α-tocopherol could reverse the cytotoxicity of GLA. The saturated fatty acid SA, which did not exhibit significant increase in lipid peroxidation, also did not induce cytotoxicity. Intracellular GSH was also determined, and there was no marked change of GSH levels in cells after incubation with GLA compared with the control. These results demonstrate that GLA could induce apoptosis in K562 cells. Apoptosis is mediated by release of cytochrome c, activation of caspase-3. Lipid peroxidation may play a role in GLA cytotoxicity.     

Interaction of caveolin-1, nitric oxide, and nitric oxide synthases in hypoxic human SK-N-MC neuroblastoma cells

Neuroblastoma cells are capable of hypoxic adaptation, but the mechanisms involved are not fully understood. We hypothesized that caveolin-1 (cav-1), a plasma membrane signal molecule, might play a role in protecting neuroblastoma cells from oxidative injury by modulating nitric oxide (NO) production. We investigated the alterations of cav-1, cav-2, nitric oxide synthases (NOS), and NO levels in human SK-N-MC neuroblastoma cells exposed to hypoxia with 2% [O2]. The major discoveries include: (i) cav-1 but not cav-2 was up-regulated in the cells exposed to 15 h of hypoxia; (ii) NO donor 1-[N, N-di-(2-aminoethyl) amino] diazen-1-ium-1, 2-diolate up-regulated the expression of cav-1, whereas the non-selective NOS inhibitor N(G)-nitro-L-arginine methyl ester and inducible NOS (iNOS) inhibitor 1400W each abolished the increase in cav-1 expression in the hypoxic SK-N-MC cells. These results suggest that iNOS-induced NO production contributes to the up-regulation of cav-1 in the hypoxic SK-N-MC cells. Furthermore, we studied the roles played by cav-1 in regulating NO, NOS, and apoptotic cell death in the SK-N-MC cells subjected to 15 h of hypoxic treatment. Both cav-1 transfection and cav-1 scaffolding domain peptide abolished the induction of iNOS, reduced the production of NO, and reduced the rates of apoptotic cell death in the hypoxic SK-N-MC cells. These results suggest that increased expression of cav-1 in response to hypoxic stimulation could prevent oxidative injury induced by reactive oxygen species. The interactions of cav-1, NO, and NOS could be an important signal pathway in protecting the neuroblastoma cells from oxidative injury, contributing to the hypoxic tolerance of neuroblastoma cells.     

Up-regulation of nitric oxide synthase by estradiol in human aorticendothelial cells

We have examined the effects of sex hormones on calcium-dependent NO production and protein levels of NO synthase in cultured human aortic endothelial cells, which were treated with various doses of 17β-estradiol and testosterone for 8–48 h. Treatment with 17β-estradiol enhanced calcium-dependent NO production, but testosterone had exerted no effect. Western blot using monoclonal anti-human endothelial NO synthase antibody clarified that increased NO production by 17β-estradiol treatment was accompanied by increased NO synthase protein. Our results provide evidence that human endothelial NO synthase can be regulated by estrogens.     

Caspase activation and cytochrome c release during HL-60 cell apoptosis induced by a nitric oxide donor

Nitric oxide (NO) from (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (NOC-18) induces apoptosis in human leukemia HL-60 cells. This effect was prevented by the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-FMK), thereby implicating caspase activity in the process. NOC-18 treatment resulted in the activation of several caspases including caspase-3, -6, -8, and -9(-like) activities and the degradation of several caspase substrates such as nuclear lamins and SP120 (hnRNP-U/SAF-A). Moreover, release of cytochrome c from mitochondria was also observed during NOC-18-induced apoptosis. This change was substantially prevented by Z-VAD-FMK, thereby suggesting that the released cytochrome c might function not only as an initiator but also as an amplifier of the caspase cascade. Bid, a death agonist member of the Bcl-2 family, was processed by caspases following exposure of cells to NOC-18, supporting the above notion. Thus, NO-mediated apoptosis in HL-60 cells involves a caspase/cytochrome c-dependent mechanism.     

Cadmium- and chromium-induced oxidative stress, DNA damage, and apoptotic cell death in cultured human chronic myelogenous leukemic K562 cells, promyelocytic leukemic HL-60 cells, and normal human peripheral blood mononuclear cells

Sodium dichromate [Cr(VI)] and cadmium chloride [Cd(II)] are both cytotoxic and mutagenic. This study examined the toxic and apoptotic potentials of these two cations on three cell types in vitro, namely, human chronic myelogenous leukemic (CML) K562 cells, promyelocytic leukemic HL-60 cells, and normal human peripheral blood mononuclear cells. The cells were incubated with 0-100 microM concentrations of the two cations for 0, 24, or 48 hours at 37 degrees C. Both Cr(VI) and Cd(II) induced changes in intracellular oxidized states of cells, which were detected using laser scanning confocal microscopy. Cell cycle modulation and apoptosis of the K562 cells by Cr(VI) and Cd(II) were determined by flow cytometry. Significant decreases in the G2/M phase were observed in the Cr(VI) and Cd(II) treated CML cells compared with untreated cells. At 12.5 microM, Cr(VI) induced greater apoptosis in K562 cells as compared with Cd(II). In the K562 cells, 2.2- and 3.0-fold increases in DNA fragmentation were observed following incubation with 12.5 and 25 microM Cr(VI), respectively, and 1.2- and 1.7-fold increases in DNA fragmentation were observed with Cd(II). Furthermore, approximately 2.7- and 4.9-fold increases in cytochrome c reduction were observed following incubation with 12.5 and 25 microM Cr(VI), respectively, and 1.6- and 3.3-fold increases in cytochrome c reduction were observed with Cd(II), demonstrating enhanced production of superoxide anion. Approximately 3.1 to 6-fold increases in hydroxyl radical production were observed following incubation of the K562 cells with these cations at 12.5 and 25 microM concentrations. These results in K562 cells were compared with promyelocytic leukemic HL-60 cells and normal human peripheral blood mononuclear cells. More pronounced effects were observed on K562 and HL-60 cells, and much lesser effects were observed on normal human peripheral blood mononuclear cells. The results demonstrate that both cations are toxic, producing oxidative tissue damage and apoptosis. Furthermore, more drastic effects were observed on K562 and HL-60 cells as compared with normal human peripheral blood mononuclear cells.     

2-D gel electrophoresis-based proteomic analysis reveals that ormeloxifen induces G0-G1 growth arrest and ERK-mediated apoptosis in chronic myeloid leukemia cells K562

Ormeloxifen is a nonsteroidal selective estrogen receptor modulator (SERM) and has been shown to possess anticancer activities in breast and uterine cancer. Here, we show that ormeloxifen induces apoptosis in dose-dependent manner in a variety of leukemia cells, more strikingly in K562. 2-DE-gel electrophoresis of K562 cells induced with ormeloxifen showed that 57 and 30% of proteins belong to apoptosis and cell-cycle pathways, respectively. Our data demonstrate that ormeloxifen-induced apoptosis in K562 cells involves activation of extracellular signal-regulated kinases (ERKs) and subsequent cytochrome c release, leading to mitochondria-mediated caspase-3 activation. Ormeloxifen-induced apoptosis via ERK activation was drastically inhibited by prior treatment of K562 cells with ERK inhibitor PD98059. Ormeloxifen also inhibits proliferation of K562 cells by blocking them in G0-G1 phase by inhibiting c-myc promoter via ormeloxifen-induced MBP-1 (c-myc promoter-binding protein) and upregulation of p21 expression. We further show that ormeloxifen-induced apoptosis in K562 is translatable to mononuclear cells isolated from chronic myeloid leukemia (CML) patients. Thus, ormeloxifen induces apoptosis in K562 cells via phosphorylation of ERK and arrests them in G0-G1 phase by reciprocal regulation of p21 and c-myc. Therefore, inclusion of ormeloxifen in the therapy of chronic myeloid leukemia can be of potential utility.     

Evaluation of some genes and proteins involved in apoptosis on human chronic myeloid leukemia cells (K562 cells) by datura innoxia leaves aqueous extract

Abstract

Datura innoxia (D. innoxia) has an extensive usage in traditional medicine and can also be used for intervention therapy in order to treat cancer. Despite of accomplishing some researches on D. innoxia mechanism, still our knowledge is very little about exact D. innoxia apoptotic mechanism on human chronic myeloid leukemia cells (K562 cells). This study purpose was to clarify the molecular mechanism of apoptosis, which was mediated by D. innoxia leaves aqueous extract in K562 cells. MTT assay and flow cytometry was applied in order to assess the viability and apoptosis induction of K562 cells and normal human lymphoid B cells in the D. innoxia presence. Finally, the expression of the apoptotic related genes (p53, BAX, BCL2, Caspases 3, 6, 7 and 9) were evaluated using quantitative Real-Time PCR. Western blot analysis was applied for assessing the protein expression. MTT results indicated that D. innoxia could inhibit the viability of K562 cells in a dose- and time-dependent manner. In parallel, D. innoxia inhibitory effect on normal human lymphoid B cells was lower in comparison with its effect on K562 cells at the same concentrations and same incubation time. Apoptosis induction in K562 cells after D. innoxia exposure was determined by flow cytometry. Apoptosis was activated by D. innoxia in K562 cells throughout increasing the expression of P53, BAX/BCL2 ratio, caspase 9, 3, 6, 7. Western blot analysis demonstrated significant increase in cleaved PARP-1 and cleaved caspase 3 in treated K562 cells with high D. innoxia leaves aqueous extract concentration. D. innoxia leaves trigger apoptosis in K562 cells throughout intrinsic apoptotic pathway.

Communicated by Ramaswamy H. Sarma     

Evidence for the role of nitric oxide in antiapoptotic and genotoxic effect of nicotine on human gingival fibroblasts

Resistance to apoptosis is essential for cancer survival and plays a critical role in carcinogenesis. Growing evidence suggests that nicotine can act as a tumor promoter, impairing apoptotic process in certain types of human cancer cell lines. Our previous study revealed in human gingival fibroblasts (HGFs) a concomitant antiapoptotic and genotoxic effect of nicotine, manifested by the attenuation of staurosporine (STP)-induced apoptosis and the increase of micronucleus frequency. The present report provides evidence that nitric oxide (NO) is critically involved in these actions. In vitro treatment with sodium nitroprusside as NO donor showed that NO produced similar effects as those observed with nicotine: it caused DNA damage and partially prevented apoptosis induced by staurosporine. Exposure of HGFs to nicotine, at concentrations similar to those found in the blood of habitual smokers, leads to the production of NO associated with the induction of inducible nitric oxide synthase (iNOS) expression. Experiments using an inhibitor of iNOS, N-monomethyl-L-arginine (NMA), together with nicotine confirmed the involvement of NO in the drug action, abrogating completely cell death and a good part of the genotoxicity. Finally, we show by different approaches that the inhibition of cell death by nicotine through NO release is related to modulation of caspase-1 activation. This work was supported by a MIUR grant to RC.     

Endotoxin elicits nitric oxide release in rat but prostacyclin synthesis in human and bovine vascular smooth muscle cells

Lipopolysaccharide (LPS) exposure to cells and tissues can mimic the biochemical events leading to septic shock. Previous data demonstrated a massive upregulation of prostaglandin endoperoxide H2 synthase (PGHS-2), but not NO synthase-2 (NOS-2) in bovine smooth muscle cells (SMC) between 2 and 12 h of LPS exposure. This caused an abundant release of prostacyclin (PGI2) by constitutive PGI2-synthase as a counterregulation to a dysfunctional endothelium. We here report that human as well as bovine SMC mainly respond by the induction of PGHS-2 and the subsequent release of PGI2, whereas rat SMC exhibited a distinct induction of NOS-2 and released significantly higher amounts of *NO compared with cattle and human. The induction of either PGHS-2 or NOS-2 in the three different species investigated seems to be mutually exclusive in the time window of 2-24 h. This finding should be considered in the setup of experimental models for the investigation of septic shock.     

Cadmium induced endothelial dysfunction: consequence of defective migratory pattern of endothelial cells in association with poor nitric oxide availability under cadmium challenge

Recent advances in cadmium toxicity research suggest an association between cadmium and vascular diseases. However, the mechanisms of cadmium implications in vascular diseases are not yet explained. The objective of our present study is to explore the mechanism of cadmium induced endothelial dysfunction. Doses of 0, 1 and 5microM cadmium chloride were used to test the effects of cadmium on nitric oxide induced tube formation, cellular migration and subcellular actin polymerization in ECV-304 endothelial cells. An egg-yolk vascular bed model was used to study the effects of cadmium on angiogenesis. Results of the present study show that 5microM cadmium chloride effectively inhibited angiogenesis, cellular migration and tube formation. Phalloidin staining, which represents actin polymerization of endothelial cells, reveals that cadmium induces an altered F-actin pattern, which could be the prime cause for cadmium mediated inhibition of cellular migration and angiogenesis. Cadmium was also found to inhibit nitric oxide production in endothelial cells in a calcium free medium, which further hints that cadmium might impair endothelial functions by inhibiting endothelial nitric oxide synthase.     

Induction of G2/M phase arrest and apoptosis by a novel enediyne derivative, THDA, in chronic myeloid leukemia (K562) cells

We studied the effect of 2-(6-(2-thieanisyl)-3(Z)-hexen-1,5-diynyl)aniline(THDA), a newly developed anti-cancer agent, on cell proliferation, cell cycle progression, and induction of apoptosis in K562 cells. THDA was found to inhibit the growth of K562 cells in a time-and dose-dependent manner. Cell cycle analysis showed G2/M phase arrest and apoptosis in K562 cells following 24 h exposure to THDA. During the G2/M arrest, cyclin-dependent kinase inhibitors (CDKIs), p21 and p27 were increased in a time-dependent manner. Analysis of the cell cycle regulatory proteins demonstrated that THDA did not change the steady-state levels of cyclin B1, cyclin D3 and Cdc25C, but decreased the protein levels of Cdk1, Cdk2 and cyclin A. THDA also caused a marked increase in apoptosis, which was associated with activation of caspase-3 and proteolytic cleavage of poly (ADP-ribose) polymerase. These molecular alterations provide an insight into THDA-caused growth inhibition, G2/M arrest and apoptotic death of K562 cells.     

Low concentration of oxidant and nitric oxide donors stimulate proliferation of human endothelial cells in vitro

Proliferation of human umbilical vein endothelial cells in vitro was inhibited by high concentrations of oxidants and nitric oxide donors but stimulated by low (micromolar or submicromolar) concentrations of hydrogen peroxide, menadione, tert-butyl hydroperoxide, AAPH, nitroglycerin, SIN-1 and sodium nitroprusside. The stimulation seems to be dependent upon generation of secondary reactive oxygen species as inferred from attenuation of cell proliferation by superoxide dismutase and catalase. These results point to another type of possible artefact of cell culture, viz. stimulation of cell proliferation by low concentrations of oxidants.