Cytotoxic activity of dimeric acridone alkaloids derived from Citrus plants towards human leukaemia HL-60 cells.

OBJECTIVES: Acridone alkaloids from Citrus and their derivatives show various kinds of biological activity. However, the anticancer activities of dimeric acridone alkaloids with unique structures and the molecular mechanism of these effects are poorly understood. METHODS: We investigated the cytotoxicity effects of dimeric acridone alkaloids isolated from Marsh grapefruit on human myeloid leukaemia HL-60 cells. KEY FINDINGS: Of the six dimeric acridone alkaloids tested, citbismine-E, the most potent, dose- and time-dependently decreased HL-60 cell viability by inducing apoptosis. The treatment of HL-60 cells with citbismine-E yielded a significant increase in levels of intracellular reactive oxygen species (ROS). Citbismine-E lowered the mitochondrial membrane potential and increased the activities of caspase-9 and -3. In addition, citbismine-E-induced apoptosis, decrease in mitochondrial membrane potential and caspase activation were significantly alleviated by pretreatment of the cells with antioxidant N-acetylcysteine (NAC). Citbismine-E induced intrinsic caspase-dependent apoptosis through ROS-mediated c-Jun N-terminal kinase activation. Citbismine-E-induced production of oxidative stress biomarkers, malondialdehyde and 8-hydroxy-2'-deoxyguanosine was also attenuated by pretreatment with NAC. CONCLUSIONS: Citbismine-E is a powerful cytotoxic agent against HL-60 cells that acts by inducing mitochondrial dysfunction-mediated apoptosis through ROS-dependent JNK activation. Citbismine-E also induced oxidative stress damage via ROS-mediated lipid peroxidation and DNA damage in HL-60 cells.

Potent apoptosis-inducing activity of erypoegin K, an isoflavone isolated from Erythrina poeppigiana, against human leukemia HL-60 cells.

Erypoegin K is an isoflavone isolated from the stem bark of Erythrina poeppigiana. It contains a furan group at the A-ring of the core isoflavone structure and can inhibit the activity of glyoxalase I, an enzyme that catalyzes the detoxification of methylglyoxal (MG), a by-product of glycolysis. In the present study, we found that erypoegin K has a potent cytotoxic effect on human leukemia HL-60 cells. Its cytotoxic effect was much stronger than that of a known glyoxalase I inhibitor S-p-bromobenzylglutathione cyclopentyl diester. Conversely, erypoegin K demonstrated weak cytotoxicity toward normal human peripheral lymphocytes. The treatment of HL-60 cells with erypoegin K significantly induced caspase-3 activity, whereas the pretreatment of the cells with caspase-3 inhibitor suppressed erypoegin K-induced cell death. Furthermore, nuclear condensation and apoptotic genome DNA fragmentation were observed in erypoegin K-treated HL-60 cells. These results indicated that the observed cell death was mediated by apoptosis. In addition, the toxic compound MG was highly accumulated in the culture medium of erypoegin K-treated HL-60 cells, suggesting that cell apoptosis was triggered by extracellular MG. The present study showed that erypoegin K has a potent apoptosis-inducing effect on cancerous cell lines, such as HL-60.

Inhibitory Effect of Isoflavones from Erythrina poeppigiana on the Growth of HL-60 Human Leukemia Cells through Inhibition of Glyoxalase I.

It has been reported that many malignant human tissues, including breast, colon, and lung cancers, may show an elevated expression of glyoxalase I (GLO I). GLO I catalyzes the reaction to transform hemimercaptal, a compound formed from methylglyoxal (MG) and reduced glutathione, into S-D-lactoylglutathione, which is then converted to D-lactic acid by glyoxalase II. GLO I inhibitors are expected to be useful for inhibiting tumorigenesis through the accumulation of apoptosis-inducible MG in tumor cells. Here, we investigated the anti-proliferative activity of eight kinds of isoflavone isolated from Erythrina poeppigiana against the growth of HL-60 human leukemia cells from the viewpoint of GLO I inhibition. Of the compounds tested, the diprenyl isoflavone, isolupalbigenin, was shown to exhibit the highest anti-proliferative activity against HL-60 cells. Upon the treatment of HL-60 cells with isolupalbigenin, MG was significantly accumulated in the culture medium, and the caspase 3 activity of the cell lysate was elevated in a time-dependent manner. Thus, it is suggested that isolupalbigenin inhibits the enzyme GLO I, resulting in MG accumulation in the medium, and leading to cell apoptosis. Isolupalbigenin, with two prenyl groups in its A- and B-rings, might be expected to become a potent leading compound for the development of anticancer agents.

Phenolic constituents from stem bark of Erythrina poeppigiana and their inhibitory activity on human glyoxalase I.

A novel isoflavone, erythgianin A (1), along with nine known compounds 2-10, was isolated from the stem bark of Erythrina poeppigiana (Leguminosae). The unusual isoflavone structure of 1, possessing a highly oxidized 3″,4″-dihydroxy-2″-hydroxymethyl-2″-methyl-2″,3″-dihydropyrano substituent, was determined on the basis of spectroscopic analyses. All of the isolated compounds were evaluated for their in vitro inhibitory activity toward human glyoxalase I. Among the isolates, isolupalbigenin (10) with two prenyl groups showed the highest inhibitory activity.

Involvement of regucalcin in lipid metabolism and diabetes.

Regucalcin (RGN/SMP30) was originally discovered in 1978 as a unique calcium-binding protein that does not contain the EF-hand motif of calcium-binding domain. The regucalcin gene (rgn) is localized on the X chromosome and is identified in over 15 species consisting the regucalcin family. Regucalcin has been shown to play a multifunctional role in cell regulation; maintaining of intracellular calcium homeostasis and suppressing of signal transduction, translational protein synthesis, nuclear deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) synthesis, proliferation, and apoptosis in many cell types. Moreover, regucalcin may play a pathophysiological role in metabolic disorder. The expression of regucalcin is stimulated through the action of insulin in liver cells in vitro and in vivo and it is decreased in the liver of rats with type I diabetes induced by streptozotocin administration in vivo. Overexpression of endogenous regucalcin stimulates glucose utilization and lipid production in liver cells with glucose supplementation in vitro. Regucalcin reveals insulin resistance in liver cells. Deficiency of regucalcin induces an impairment of glucose tolerance and lipid accumulation in the liver of mice in vivo. Overexpression of endogenous regucalcin has been shown to decrease triglyceride, total cholesterol and glycogen contents in the liver of rats, inducing hyperlipidemia. Leptin and adiponectin mRNA expressions in the liver tissues are decreased in regucalcin transgenic rats. Decrease in hepatic regucalcin is associated with the development and progression of nonalcoholic fatty liver disease and fibrosis in human patients. Regucalcin may be a key molecule in lipid metabolic disorder and diabetes.

Rotenoid derivatives from Derris trifoliata with nitric oxide production inhibitory activity.

Study of the chemical constituents of the stems of Derris trifoliata Lour. (Leguminosae) collected in Singapore led to the isolation and identification of three known and two new rotenoid derivatives. The new derivatives, named derrisfolin A (1) and B (2), inhibited nitric oxide production in murine macrophage-like RAW 264.7 cells stimulated with interferon-gamma and lipopolysaccharide.

Apoptosis of HL-60 leukemia cells induced by carbazole alkaloids isolated from Murraya euchrestifolia.

We carried out primary screening of 13 carbazole alkaloids isolated from the plant species Murraya euchrestifolia (Rutaceae) on cell growth inhibition of the human leukemia cell line HL-60. Among them, murrayafoline-A (1) and murrayazolinine (7) exhibited significant growth suppression due to apoptosis mediated by the activation of the caspase-9/caspase-3 pathway.

Methyl galbanate, a novel inhibitor of nitric oxide production in mouse macrophage RAW264.7 cells.

It is well known that inflammation is associated with various neurodegenerative diseases, such as Parkinson's disease and Alzheimer's disease. An inflammatory mediator, nitric oxide (NO), is produced by inducible NO synthase (iNOS) in microglia and seems to be one of the possible causes of neurodegeneration. Several natural and synthetic compounds which exert anti-inflammatory effects by inhibiting NO production have been reported to date. The aim of this work was to investigate whether any of the 6 terpenoid coumarins (methyl galbanate, galbanic acid, farnesiferol A, badrakemone, umbelliprenin, and aurapten) isolated from Ferula szowitsiana DC. have inhibitory activity against NO production in RAW264.7 mouse macrophage cells stimulated with lipopolysaccharide (LPS) and interferon-γ (IFN-γ). Of the 6 terpenoid coumarins tested, methyl galbanate significantly decreased NO production in LPS/IFN-γ-stimulated RAW264.7 cells. In the presence of methyl galbanate, LPS/IFN-γ-induced iNOS mRNA expression was significantly decreased to 52% of the level found with LPS/IFN-γ stimulation alone. Methyl galbanate slightly attenuated COX-2 mRNA expression. Using the RAW264.7-tsAM5NE co-culture system, we showed that methyl galbanate protected neuronally differentiated tsAM5NE cells from NO-induced cell death by inhibiting the production of NO. Our finding suggests that methyl galbanate may be useful for developing a new drug against neurodegenerative diseases.

Induction of apoptosis by isoflavonoids from the leaves of Millettia taiwaniana in human leukemia HL-60 cells.

We have isolated two new isoflavonoids, millewanin-F (1) and furowanin-A (2), together with five known isoflavonoids from the leaves of Millettia taiwaniana Hayata (Leguminosae) and examined their effects on the growth of human leukemia HL-60 cells. Among the isolated isoflavonoids, furowanin-A (2), warangalone (3), isoerysenegalensein-E (4), and euchrenone b10 (6) showed significant cytotoxicity against HL-60 cells. After treatment with three of the cytotoxic isoflavonoids, furowanin-A (2), warangalone (3), and isoerysenegalensein-E (4), fluorescence microscopy with Hoechst 33,342 staining revealed that the percentage of apoptotic cells with fragmented nuclei and condensed chromatin increased in a time-dependent manner. In addition, the activities of caspase-9 and caspase-3 were also enhanced in a time-dependent manner upon treatment with the isoflavonoids 2, 3, and 4. Caspase-9 and caspase-3 inhibitors suppressed apoptosis induced by isoflavonoids 2, 3, and 4. These results suggest that the isoflavonoids induced apoptosis in HL-60 cells through activation of the caspase-9/caspase-3 pathway, which is triggered by mitochondrial dysfunction.

Recruitment of mRNA-destabilizing protein TIS11 to stress granules is mediated by its zinc finger domain.

TIS11, a member of the CCCH zinc finger protein family, was found to be distributed throughout cells with a preferential cytoplasmic localization when transiently expressed in COS-7 cells. Upon treatment with heat shock, TIS11 became localized in discrete particles in the cytoplasm of the transfectants. We showed the TIS11-positive particles to be stress granules (SGs), which are known to be formed in the cytoplasm of eukaryotic cells in response to environmental stresses. By deletion studies using the green fluorescent protein fusion system, we mapped a functional stress granule (SG) localization signal to a region containing two tandem repeats of the zinc finger motif of TIS11. Site-directed mutations of Tyr105/Tyr113, Gly109/Gly 114, and Phe119 in the first zinc finger motif diminished the ability of this TIS11 domain to direct SG localization. Importantly, when the zinc-chelating Cys residues in either the first or second zinc finger were mutated to Ala residues, the recruitment of the TIS11 zinc finger region to SG was significantly inhibited by the mutation and was completely abolished by the mutation in both zinc fingers. These results suggest that recruitment of TIS11 to heat shock-induced SG is governed by the tandem zinc finger domains of this protein.

Quinolone alkaloids with nitric oxide production inhibitory activity from Orixa japonica.

Four new quinolone alkaloids, orixalone A (1), B (2), C (3), and D (4), together with 12 known compounds were isolated from the stems of Orixa japonica. Orixalone A (1) inhibited nitric oxide production in murine macrophage-like RAW 264.7 cells stimulated with interferon-gamma and lipopolysaccharide.

Identification of nuclear import and export signals within the structure of the zinc finger protein TIS11.

TIS11, a member of the CCCH zinc finger protein family, functions as a positive transcriptional regulator. TIS11 was localized in both the cytoplasm and nucleus when transiently expressed in COS-7 cells. Upon treatment with leptomycin B, a specific inhibitor of the nuclear export receptor CRM1, a marked nuclear accumulation of TIS11 was observed, indicating that TIS11 shuttles between the nucleus and the cytoplasm. By deletion studies using a green fluorescent protein fusion system, we have mapped a functional nuclear localization signal (NLS) to a region containing two tandem repeats of the zinc finger motif of TIS11. A site-directed mutagenesis analysis of TIS11 NLS has revealed the critical importance of two arginine residues (Arg127 and Arg131 in the rat TIS11). Furthermore, we demonstrated that the N-terminal Leu-rich region of TIS11 serves as an LMB-sensitive nuclear export signal (NES), indicating that TIS11 follows a CRM1-mediated export pathway. These results suggest that TIS11 is subject to constant nucleocytoplasmic shuttling due to its NLS and NES.