Induction of apoptosis by three marine algae through generation of reactive oxygen species in human leukemic cell lines

In this study, we examined the antitumor effect of marine algae extracts on human hepatoma and leukemia cells. Ethyl acetate extracts from Colpomenia sinuosa (Cs-EA), Halimeda discoidae (Hd-EA), and Galaxaura oblongata (Go-EA) directly inhibited the growth of human hepatoma HuH-7 cells and leukemia U937 and HL-60 cells in a time- and dose-dependent manner. Specifically, these algae extracts induced apoptosis of U937 and HL-60 cells as evaluated by detection of hypodiploid cells using flow cytometry and observation of condensed and fragmented nuclei in algae extract-treated cells. Intracellular reactive oxygen species (ROS), especially hydrogen peroxide and superoxide anion, were increased about 2-3-fold in U937 cells treated with Cs-EA for 3-5 h. Interestingly, antioxidant N-acetylcysteine effectively blocked Cs-EA-, Hd-EA-, and Go-EA-induced apoptosis, suggesting that ROS is a key mediator in the apoptotic signaling pathway. In conclusion, our results show that algae extracts induce apoptosis in human leukemia cells through generation of ROS.     

5-Hydroxy-3,6,7,8,3',4'-hexamethoxyflavone induces apoptosis through reactive oxygen species production, growth arrest and DNA damage-inducible gene 153 expression, and caspase activation in human leukemia cells

This study examined the growth inhibitory effects of structurally related polymethoxylated flavones in human cancer cells. Here, we report that 5-hydroxy-3,6,7,8,3',4'-hexamethoxyflavone (5-OH-HxMF) induces growth inhibition of human cancer cells and induction of apoptosis in HL-60 cells through modulation of mitochondrial functions regulated by reactive oxygen species (ROS). ROS generation occurs in the early stages of 5-OH-HxMF-induced apoptosis, preceding cytochrome c release, caspase activation, and DNA fragmentation. The changes occurred after single breaks in DNA were detected, suggesting that 5-OH-HxMF induced irreparable DNA damage, which in turn triggered the process of apoptosis. Up-regulation of Bax was found in 5-OH-HxMF-treated HL-60 cells. In addition, a caspase-independent pathway indicated by endonuclease G also contributed to apoptosis caused by 5-OH-HxMF. Antioxidants suppress 5-OH-HxMF-induced apoptosis. 5-OH-HxMF markedly enhanced growth arrest DNA damage-inducible gene 153 (GADD153) protein in a time-dependent manner. N-acetylcysteine (NAC) and catalase prevented up-regulation of GADD153 expression caused by 5-OH-HxMF. These findings suggest that 5-OH-HxMF creates an oxidative cellular environment that induces DNA damage and GADD153 gene activation, which in turn helps trigger apoptosis in HL-60 cells. Meanwhile, ROS were proven an important inducer in this apoptotic process. The C-5 hydroxyl on the ring of 5-OH-HxMF was found to be essential for the antiproliferative and apoptosis-inducing activity. Our study identified the novel mechanisms of 5-OH-HxMF-induced apoptosis and indicated that these results have significant applications as potential chemopreventive and chemotherapeutic agents.     

Induction of apoptosis by 1-(2-hydroxy-5-methylphenyl)-3-phenyl-1,3-propanedione through reactive oxygen species production, GADD153 expression, and caspases activation in human epidermoid carcinoma cells

This study examined the growth inhibitory effects of the structurally related beta-diketones compounds in human cancer cells. Here, we report that 1-(2-hydroxy-5-methylphenyl)-3-phenyl-1,3-propanedione (HMDB) induces growth inhibition of human cancer cells and induction of apoptosis in A431 cells through modulation of mitochondrial functions regulated by reactive oxygen species (ROS). ROS generation occurs in the early stages of HMDB-induced apoptosis, preceding cytochrome c release, caspase activation, and DNA fragmentation. The changes occurred after single breaks in DNA were detected, suggesting that HMDB induced irreparable DNA damage, which in turn triggered the process of apoptosis. Up-regulation of Bad and p21; down-regulation of Bcl-2, Bcl-XL, Bid, p53, and fatty acid synthase; and cleavage of Bax were found in HMDB-treated A431 cells. Glutathione and N-acetylcysteine (NAC) suppress HMDB-induced apoptosis. HMDB markedly enhanced growth arrest DNA damage inducible gene 153 (GADD153) mRNA and protein in a time- and concentration-dependent manner. NAC prevented up-regulation of GADD153 mRNA expression caused by HMDB. These findings suggest that HMDB creates an oxidative cellular environment that induces DNA damage and GADD153 gene activation, which in turn helps trigger apoptosis in A431 cells.     

Induction of apoptosis by garcinol and curcumin through cytochrome c release and activation of caspases in human leukemia HL-60 cells

Garcinol, a polyisoprenylated benzophenone, was purified from Garcinia indica fruit rind. The effects of garcinol and curcumin on cell viability in human leukemia HL-60 cells were investigated. Garcinol and curcumin displayed strong growth inhibitory effects against human leukemia HL-60 cells, with estimated IC(50) values of 9.42 and 19.5 microM, respectively. Garcinol was able to induce apoptosis in a concentration- and time-dependent manner; however, curcumin was less effective. Treatment with garcinol caused induction of caspase-3/CPP32 activity in a dose- and time-dependent manner, but not caspase-1 activity, and induced the degradation of poly(ADP-ribose) polymerase (PARP). Pretreatment with caspase-3 inhibitor inhibited garcinol-induced DNA fragmentation. Treatment with garcinol (20 microM) caused a rapid loss of mitochondrial transmembrane potential, release of mitochondrial cytochrome c into cytosol, and subsequent induction of procaspase-9 processing. The cleavage of D4-GDI, an abundant hematopoietic cell GDP dissociation inhibitor for the Ras-related Rho family GTPases, occurred simultaneously with the activation of caspase-3 but preceded DNA fragmentation and the morphological changes associated with apoptotic cell death. Of these, Bcl-2, Bad, and Bax were studied. The level of expression of Bcl-2 slightly decreased, while the levels of Bad and Bax were dramatically increased in cells treated with garcinol. These results indicate that garcinol allows caspase-activated deoxyribonuclease to enter the nucleus and degrade chromosomal DNA and induces DFF-45 (DNA fragmentation factor) degradation. It is suggested that garcinol-induced apoptosis is triggered by the release of cytochrome c into the cytosol, procaspase-9 processing, activation of caspase-3 and caspase-2, degradation of PARP, and DNA fragmentation caused by the caspase-activated deoxyribonuclease through the digestion of DFF-45. The induction of apoptosis by garcinol may provide a pivotal mechanism for its cancer chemopreventive action.     

Induction of apoptosis by the Lactuca indica L. in human leukemia cell line and its active components

Lactuca indica L. (Compositae family) is used as a folk medicine in anti-inflammatory, antibacterial, antidiabetic, and other medications in Asia. The objectives of this study were to evaluate the antiproliferative effect of ethanol extracts of Lactuca indica L. (EEL) on human leukemic HL-60 cell lines and its active components. The results showed that EEL exhibited strong cytotoxic effects against HL-60 cells; the IC50 value was 313 microg/mL. Flow cytometric analysis of the externalization of phosphatidylserine (PS) using the annexin V/PI method on EEL-treated HL-60 cells showed a concentration-dependent increase of apoptosis. Moreover, EEL could induce typical DNA fragmentation in a concentration- and time-dependent manner as determined by electrophoresis and TUNEL assays. The treatment of HL-60 cells with EEL induced significant accumulation of cells in the G0/G1 phase, indicating that EEL is a cell-cycle-dependent anticancer agent. Our results also indicate that EEL-induced apoptosis in HL-60 cells is associated with the loss of mitochondrial membrane potential (delta psi m). EEL contains 5% phenolic compounds, such as quercetin, caffeic acid, rutin, and chlorogenic acid. Among the four active phenolic compounds, quercetin was found to be the most effective in inhibition against cell viability and in alteration of mitochondrial function. Our results suggest that the induction of apoptosis by EEL might offer a pivotal mechanism for its chemopreventive action.     

Selective growth inhibition of human leukemia and human lymphoblastoid cells by resveratrol via cell cycle arrest and apoptosis induction

There is great interest in the potential chemopreventive activity of resveratrol against human cancers. However, there are conflicting results on its growth inhibitory effect on normal cells. This project examined the differential effect of resveratrol at physiologically relevant concentrations on nonmalignant (WIL2-NS) and malignant (HL-60) cell lines and compared the underlying mechanisms via cell cycle modulation, apoptosis induction, and genotoxicity potential. Twenty-four hours of exposure to resveratrol was toxic to WIL2-NS and HL-60 cells in a dose-dependent manner. WIL2-NS cells regrew 5 times more than HL-60 cells by 120 h after the removal of 100 microM resveratrol (p < 0.05). Furthermore, significant alterations in cell cycle kinetics were induced by resveratrol in HL-60 cells, but were to a lesser extent for WIL2-NS cells. The proportion of apoptosis was also 3 times higher in HL-60 cells as compared to WIL2-NS cells for 100 microM resveratrol (p < 0.05). In conclusion, resveratrol preferentially inhibited the growth of HL-60 cells via cell cycle modulation and apoptosis induction and subsequently directed the cells to irreversible cell death, whereas the effect on WIL2-NS cells was largely reversible.     

Bioavailability and efficacy of vitamin D2 from UV-irradiated yeast in growing, vitamin D-deficient rats

New food sources are needed to bridge the gap between vitamin D intake and recommended intake. We assessed the bioavailability and efficacy of vitamin D in an 8 week dose-response study of bread made with vitamin D2-rich yeast compared to vitamin D3 in growing, vitamin D-deficient rats. Plasma 25-hydroxyvitamin D (25OHD) levels increased in a curvilinear, dose-dependent manner with both forms of vitamin D, but rats fed vitamin D2-rich yeast achieved lower levels than rats fed vitamin D3. Rats fed the highest doses of vitamin D had significantly greater (p<0.05) trabecular BMC, BMD, bone volume, and connectivity density, and greater midshaft total cross-sectional area, compared to rats on the vitamin D-deficient diets, with no significant difference due to vitamin D source. Vitamin D2-rich yeast baked into bread is bioavailable and improves bone quality in vitamin D-deficient animals.     

The effects of mitogen-activated protein kinase inhibitors or small interfering RNAs on gallic acid-induced HeLa cell death in relation to reactive oxygen species and glutathione

Gallic acid (GA) is widely distributed in various plants and foods and has various biological properties including anticancer effects. In this study, we investigated the effects of mitogen-activated protein kinase (MAPK) [MAP 20 kinase or ERK kinase (MEK), c-Jun N-terminal kinase (JNK), or p38)] inhibitors or small interfering RNAs (siRNAs) on GA-induced HeLa cell death in relation to reactive oxygen species (ROS) and glutathione (GSH) levels. GA dose dependently inhibited the growth of HeLa cells via apoptosis and/or necrosis at 24 h, which was accompanied by the loss of mitochondrial membrane potential (MMP; ΔΨ(m)). Treatment with 70 μM GA increased the ROS level including O(2)(?-) and significantly induced GSH depletion in HeLa cells. GA decreased the activity of extracellular signal-regulated kinase (ERK) at 24 h, whereas it increased that of JNK at the same time. While the MEK inhibitor or ERK siRNA did not affect cell growth and death in 70 μM GA-treated HeLa cells at 24 h, JNK and p38 inhibitors enhanced cell growth inhibition and death in these cells. Additionally, p38 siRNA administration augmented growth inhibition, death, and MMP (ΔΨ(m)) loss in 70 μM GA-treated HeLa cells. In relation to ROS and GSH levels, JNK and p38 inhibitors increased ROS levels, and GSH-depleted cell numbers in GA-treated HeLa cells. Moreover, p38 siRNA increased O(2)(?-) levels and GSH depletion in GA-treated HeLa cells. Each MAPK inhibitor and siRNA differentially affected ROS and GSH levels in HeLa control cells. Conclusively, JNK and p38 inhibitors and p38 siRNA enhanced growth inhibition and cell death in GA-treated HeLa cells, which were to some extent related to GSH depletion and ROS levels, especially O(2)(?-).     

Benzyl isothiocyanate (BITC) induces G2/M phase arrest and apoptosis in human melanoma A375.S2 cells through reactive oxygen species (ROS) and both mitochondria-dependent and death receptor-mediated multiple signaling pathways

Benzyl isothiocyanates (BITC), a member of the isothiocyanate (ITC) family, inhibits cell growth and induces apoptosis in many types of human cancer cell lines. The present study investigated mechanisms underlying BITC-induced apoptosis in A375.S2 human melanoma cancer cells. To observe cell morphological changes and viability, flow cytometric assays, cell counting, and a contrast-phase microscopic examination were carried out in A375.S2 cells after BITC treatment. Cell cycle distribution and apoptosis were assessed with the analysis of cell cycle by flow cytometric assays, DAPI staining, propidium iodide (PI), and annexin V staining. Apoptosis-associated factors such as reactive oxygen species (ROS) formation, loss of mitochondrial membrane potential (ΔΨ(m)), intracellular Ca(2+) release, and caspase-3 activity were evaluated by flow cytometric assays. Abundance of cell cycle and apoptosis associated proteins was determined by Western blotting. AIF and Endo G expression was examined by confocal laser microscope. Results indicated that (1) BITC significantly reduced cell number and induced cell morphological changes in a dose-dependent manner in A375.S2 cells; (2) BITC induced arrest in cell cycle progression at G(2)/M phase through cyclin A, CDK1, CDC25C/Wee1-mediated pathways; (3) BITC induced apoptosis and increased sub-G(1) population; and (4) BITC promoted the production of ROS and Ca(2+) and loss of ΔΨ(m) and caspase-3 activity. Furthermore, BITC induced the down-regulation of Bcl-2 expression and induced up-regulation of Bax in A375.S2 cells. Moreover, BITC-induced cell death was decreased after pretreatment with N-acetyl-l-cysteine (NAC, a ROS scavenger) in A375.S2 cells. In conclusion, the results showed that BITC promoted the induction of G(2)/M phase arrest and apoptosis in A375.S2 human melanoma cells through ER stress- and mitochondria-dependent and death receptor-mediated multiple signaling pathways. These data suggest that BITC has potential as an agent for the treatment of melanoma.     

Induction of autophagy and apoptosis by the extract of Solanum nigrum Linn in HepG2 cells

Solanum nigrum L. (SN) has been used in traditional folk medicine to treat different cancers. It is also used as a hepatoprotective and anti-inflammatory agent. In this study, we demonstrated that the extract of SN (SNE) induced a strong cytotoxic effect toward HepG2 cells but much less to Chang liver and WRL-68 cells. The mechanisms of the cytotoxic effect were concentration-dependent. High doses of SNE (2 and 5 mg/mL) induced apoptotic cell death in HepG2 cells, as evidenced by increases in the expressions of p-JNK and Bax, mitochodrial release of cytochrome c, and caspase activation. On the other hand, cells treated with low concentrations of SNE (50-1000 microg/mL) revealed morphological and ultrastructural changes of autophagocytic death under electron microscopic observation. Furthermore, these cells showed increased levels of autophagic vacuoles and LC3-I and LC3-II proteins, specific markers of autophagy. The levels of Bcl-2 and Akt that have been implicated in the down-regulation of autophagy were decreased upon SNE treatment. Taken together, these findings indicate that SNE induced cell death in hepatoma cells via two distinct antineoplastic activities of SNE, the ability to induce apoptosis and autophagocytosis, therefore suggesting that it may provide leverage to treat liver cancer.     

Inhibition of xanthine oxidase and suppression of intracellular reactive oxygen species in HL-60 cells by theaflavin-3,3'-digallate, (-)-epigallocatechin-3-gallate, and propyl gallate

The inhibitory effects of five tea polyphenols, namely theaflavin (TF1), theaflavin-3-gallate (TF2), theaflavin-3,3'-digallate (TF3), (-)-epigallocatechin-3-gallate (EGCG), and gallic acid, and propyl gallate (PG) on xanthine oxidase (XO) were investigated. These six antioxidant compounds reduce oxidative stress. Theaflavins and EGCG inhibit XO to produce uric acid and also act as scanvengers of superoxide. TF3 acts as a competitive inhibitor and is the most potent inhibitor of XO among these compounds. Tea polyphenols and PG all have potent inhibitory effects (>50%) on PMA-stimulated superoxide production at 20 approximately 50 microM in HL-60 cells. Gallic acid (GA) showed no inhibition under the same conditions. At 10 microM, only EGCG, TF3, and PG showed significant inhibition with potency of PG > EGCG > TF3. The superoxide scavenging abilities of these six compunds are as follows: EGCG > TF2 > TF1 > GA > TF3 > PG. PG was the most potent inhibitor of PMA-stimulated H(2)O(2) production in HL-60 cells. The order of H(2)O(2) scavenging ability was TF2 > TF3 > TF1 > EGCG > PG > GA. Therefore, the antioxidative activity of tea polyphenols and PG is due not only to their ability to scavenge superoxides but also to their ability to block XO and related oxidative signal transducers.     

Detection and quantification of provitamin D2 and vitamin D2 in hop (Humulus lupulus L.) by liquid chromatography-diode array detection-electrospray ionization tandem mass spectrometry

In this work, ergosterol and ergocalciferol were identified for the first time in hop. In addition, in this article, a simple and reliable analytical methodology for analysis of these compounds in different commercial forms of hop is presented. The performance of the method was assessed by the evaluation of parameters such as absolute recovery (higher than 70%), repeatability (lower than 3 %), linearity ( r(2) > 0.9988) and limits of detection (ranging from 0.034 for ergocalciferol to 0.058 mg/L for ergosterol) and quantification (ranging from 0.113 for ergocalciferol to 0.195 mg/L for ergosterol). On the basis of standard additions applied with the optimized procedure and high-performance liquid chromatography with diode array detection, it appears that the Nugget hop plant (crop 2006) contains 1.84 +/- 0.09 microg/g of ergosterol and 1.95 +/- 0.05 microg/g of ergocalciferol. The identity of the compounds was confirmed by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry in the positive ion mode. The presence of ergosterol here reported should have great potential for the assessment of hop as related to the fungal contamination proportion and hence the quality of this raw material.     

Isoobtusilactone A sensitizes human hepatoma Hep G2 cells to TRAIL-induced apoptosis via ROS and CHOP-mediated up-regulation of DR5

Hepatoma cells are relatively resistant to TRAIL. We have previously shown that isoobtusilactone A (IOA), a potent anticancer agent isolated from Cinnamomum kotoense, induced mitochondria-mediated apoptosis in hepatoma cells. Here, we report that IOA could potentiate TRAIL-induced apoptosis in Hep G2 cells. The combined treatment with IOA and TRAIL significantly induced caspase-dependent apoptosis. This correlated with the up-regulation of C/EBP homologous protein (CHOP) and death receptor 5 (DR5) protein levels. Gene silencing of the DR5 by small interfering RNA abrogated the apoptosis induced by the combined regimen of IOA and TRAIL, suggesting that the sensitization to TRAIL was mediated through DR5. By analyzing the DR5 promoter, we found that IOA induced a CHOP-dependent DR5 transactivation. DR5 expression after IOA treatment was accompanied by provoking intracellular reactive oxygen species (ROS) generation. Pretreatment with N-acetyl-L-cysteine (NAC) attenuated IOA-induced CHOP and DR5 expression and inhibited TRAIL-induced apoptosis. Taken together, our data suggested that ROS-dependent and CHOP-regulated DR5 expression played a pivotal role in the synergistic enhancement of TRAIL-induced apoptosis instigated by IOA in Hep G2 cells.     

Induction of apoptosis in cancer cells by Bilberry (Vaccinium myrtillus) and the anthocyanins

Among ethanol extracts of 10 edible berries, bilberry extract was found to be the most effective at inhibiting the growth of HL60 human leukemia cells and HCT116 human colon carcinoma cells in vitro. Bilberry extract induced apoptotic cell bodies and nucleosomal DNA fragmentation in HL60 cells. The proportion of apoptotic cells induced by bilberry extract in HCT116 was much lower than that in HL60 cells, and DNA fragmentation was not induced in the former. Of the extracts tested, that from bilberry contained the largest amounts of phenolic compounds, including anthocyanins, and showed the greatest 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity. Pure delphinidin and malvidin, like the glycosides isolated from the bilberry extract, induced apoptosis in HL60 cells. These results indicate that the bilberry extract and the anthocyanins, bearing delphinidin or malvidin as the aglycon, inhibit the growth of HL60 cells through the induction of apoptosis. Only pure delphinidin and the glycoside isolated from the bilberry extract, but not malvidin and the glycoside, inhibited the growth of HCT116 cells.     

High-pressure liquid chromatographic determination of vitamin D3 in resins, oils, dry concentrates, and dry concentrates containing vitamin A.

A high-pressur liquid chromatographic (HPLC) procedure has been developed for the determination of vitamin D3 in resins, oils, dry concentrates, and dry concentrates containing vitamin A. Specificity of the method for vitamin D3 in the presence of isomers and other related constituents was shown by ultimate recovery of the vitamin D3 and the individual constituents and their characterization by other methods such as ultraviolet spectrophotometry. Precision and accuracy are within 2%, and as many as 20 determinations may be completed in a working day. Excellent agreement with the official AOAC biological assay was found. A comparison of the response of isomers of vitamin D3 by the HPLC method vs. other instrumental and chemical procedures shows HPLC to be the most specific method for determining the bioactive isomers.     

Inhibition of reactive nitrogen species effects in vitro and in vivo by isoflavones and soy-based food extracts

Recent studies have shown that soy isoflavone inhibits inducible nitric oxide (NO) synthase activities and is reported to have peroxynitrite scavenging ability. Consequently, we investigated whether isoflavones (daidzein and genistein) and extracts from soy-based products (miso, soymilk, tofu, soy sprout, black soybean, soybean, and yuba) would inhibit the reactive nitrogen species (RNS) effect in vitro and in vivo. In the in vitro experiments [including the protection of cellular DNA from peroxynitrite or sodium nitroprusside damage, an inhibitory effect on nitric oxide production from lipopolysaccharide (LPS)-induced RAW 264.7 cells, and nitric oxide scavenging ability], extracts from soy-based foods showed a potent antioxidant activity and an inhibiting effect on RNS activity. These effects were correlated with total isoflavone content. In the in vivo experiments, rats were given isoflavones (4.0 mg/kg bw) or soy-based product extracts (1.0 g/kg bw) orally for 1 week and were injected with vehicle H(2)O (1 mL/kg bw) or LPS (10 mg/kg bw) on the day 7. Twelve hours after treatment, the rats were killed, and blood serum was collected for analysis. The intraperitoneal administration of LPS resulted in an increase in serum nitrite, nitrate, and nitrotyrosine concentrations. These are stable metabolite end products of nitric oxide, to 4-, 16-, and 5-fold levels, (4, 10 microM and 58 +/- 14 pmol/mL), of the placebo control, respectively. Results showed that oral administration of isoflavones and extracts from soy-based products significantly decreased serum nitrite, nitrate, and nitrotyrosine levels in LPS-induced rats. This study demonstrates that soy isoflavone supplementation may inhibit RNS-induced oxidation both in vitro and in vivo.     

Levels of active oxygen species are controlled by ascorbic acid and anthocyanin in Arabidopsis

Stabilization of the levels of active oxygen species (AOS) is important to the survival of organisms. To clarify the system controlling levels of AOS in plants, this study used an electron spin resonance (ESR) method to directly measure superoxide radical (O(2)(.-)) scavenging activities in the wild-type Arabidopsis thaliana (Col and Ler ecotypes), two anthocyanin mutants (tt3 and ttg1), and an ascorbic acid mutant (vtc1). Under ordinary growth conditions, Arabidopsis contained superoxide-scavenging activity (SOSA) of approximately 300-500 SOD units/g of fresh weight. The ESR pattern indicated that most (40-50%) of this activity was due to ascorbic acid. For the analysis of SOSA under conditions of oxidative stress, synthesis of AOS was induced by gamma-irradiation. The radical scavenging activity in irradiated plants increased approximately 10-fold following an associated increase in the accumulation of ascorbic acid and anthocyanin. The accumulation of ascorbic acid and anthocyanin was suppressed by treatment with an antioxidant before irradiation and was induced by treatment with a radical-generating reagent. The contributions of ascorbic acid and anthocyanin to the total superoxide radical scavenging activity differed among ecotypes. In the Ler ecotype, ascorbic acid accumulated at twice the level of that in the Col ecotype, and induction of anthocyanin was half that in Col. To confirm the activity of ascorbic acid and anthocyanin against AOS stress, the viability of the wild type and mutants (tt2, tt3,tt5, ttg1, and vtc1) was examined after gamma-irradiation. Only the plants in which ascorbic acid and anthocyanin were induced had the ability to grow and flower.     

Cyanidin and Malvidin from Oryza sativa cv. Heugjinjubyeo mediate cytotoxicity against human monocytic leukemia cells by arrest of G(2)/M phase and induction of apoptosis

Oryza sativa cv. Heugjinjubyeo (Gramineae), anthocyanin-pigmented rice, having dark purple grains, is known broadly as enriched rice with an improved taste. Two bioactive compounds were isolated from the 0.5% HCl-ethyl alcohol soluble fraction of the aleurone layer of O. sativa cv. Heugjinjubyeo through an activity-monitored fractionation and isolation method. From spectral analysis, the cytotoxic components were the anthocyanidins cyanidin (1) and malvidin (2) The 50% growth inhibitory concentrations (IC(50)) of cyanidin and malvidin on U937, human monocytic leukemia cells, were 60 and 40 microg/mL, respectively. These compounds showed cytotoxicity through the arrest of the G(2)/M phase of cell cycle and induction of apoptosis.     

Plants as biofactories: physiological role of reactive oxygen species on the accumulation of phenolic antioxidants in carrot tissue under wounding and hyperoxia stress

Plants subjected to postharvest abiotic stresses synthesize secondary metabolites with health-promoting properties. Here, we report the potential use of carrots (Daucus carota) as biofactories of caffeoylquinic acids when subjected to wounding and hyperoxia stresses. Wounding stress induced an increase of ～287% in total phenolic content (PC) in carrots stored for 48 h at 20 °C. This increase was higher (～349%) in the wounded tissue treated with hyperoxia stress. To further understand the physiological role of reactive oxygen species (ROS) as a signaling molecule for the stress-induced accumulation of phenolics in carrots, the respiration rate as well as the enzymatic activities of NADPH oxidase, superoxide dismutase, ascorbate peroxidase, and catalase were evaluated. Likewise, shredded carrots were treated with diphenyleneiodonium chloride solution to block NADPH oxidase ROS productions, and the phenylalanine ammonia lyase activity and total PC were evaluated. Results demonstrated that ROS play a key role as a signaling molecule for the stress-induced accumulation of PC in carrots.     

Exposure of coelomocytes from the earthworm Eisenia hortensis to Cu,Cd, and dimethylbenz[a]anthracene: An in vitro study examining reactive oxygen species production and immune response inhibition

The principal aims of this study were to investigate the in vitro effects of copper (Cu), cadmium (Cd) and the polycyclic aromatic hydrocarbon (PAH) dimethylbenz[a]anthracene (DMBA) on reactive oxygen species (ROS) production and the effect of DMBA on immune function (phagocytosis) in coelomocyte samples isolated from the earthworm Eisenia hortensis. Using a fluorogenic indicator of ROS, dihydrorhodamine 123, we show that earthworm coelomocytes are induced to produce significant increases in ROS when exposed to Cu or Cd at 32–250 μM, an effect diminished by the chelator ethylenediaminetetraacetic acid (EDTA). DMBA also induced significant ROS production in the range of 20–400 μM compared to controls. Testing the effect of DMBA on cellular immune mechanisms, we used a phagocytosis assay to reveal an immunosuppressive effect of DMBA (50–200 μM) on the capacity of hyaline amoebocytes to ingest fluorescent Escherichia coli. These studies illustrate the relative ease and efficiency of these in vitro procedures for testing the toxicological effects of environmental pollutants, including heavy metals and PAHs, using earthworm coelomocytes as indicators of oxidative stress and immune response impairment.