Novel roles of folic acid as redox regulator: Modulation of reactive oxygen species sinker protein expression and maintenance of mitochondrial redox homeostasis on hepatocellular carcinoma.

We provide herein several lines of evidence to substantiate that folic acid (or folate) is a micronutrient capable of functioning as a novel redox regulator on hepatocellular carcinoma. First, we uncovered that folate deficiency could profoundly downregulate two prominent anti-apoptotic effectors including survivin and glucose-regulated protein-78. Silencing of either survivin or glucose-regulated protein-78 via small interfering RNA interfering technique established that both effectors could serve as reactive oxygen species sinker proteins. Second, folate deficiency-triggered oxidative-nitrosative stress could strongly induce endoplasmic reticulum stress that in turn could provoke cellular glutathione depletion through the modulation of the following two crucial events: (1) folate deficiency could strongly inhibit Bcl-2 expression leading to severe suppression of the mitochondrial glutathione pool and (2) folate deficiency could also profoundly inhibit two key enzymes that governing cellular glutathione redox regulation including γ-glutamylcysteinyl synthetase heavy chain, a catalytic enzyme for glutathione biosynthesis, and mitochondrial isocitrate dehydrogenase 2, an enzyme responsible for providing nicotinamide adenine dinucleotide phosphate necessary for regenerating oxidized glutathione disulfide back to glutathione via mitochondrial glutathione reductase. Collectively, we add to the literature new data to strengthen the notion that folate is an essential micronutrient that confers a novel role to combat reactive oxygen species insults and thus serves as a redox regulator via upregulating reactive oxygen species sinker proteins and averting mitochondrial glutathione depletion through proper maintenance of redox homeostasis via positively regulating glutathione biosynthesis, glutathione transporting system, and mitochondrial glutathione recycling process.

Survivin-mediated cancer cell migration through GRP78 and epithelial-mesenchymal transition (EMT) marker expression in Mahlavu cells.

BACKGROUND: Survivin has multiple functions during the progression of cancer. However, the role of survivin in the progression and metastasis of hepatocellular carcinoma (HCC) remains unknown. MATERIALS AND METHODS: Survivin expression in HCC cells (Mahlavu and Hep3B) was assessed using reverse transcription real-time PCR and Western blot analyses. In addition, survivin expression in HCC cells was manipulated using small interfering RNA (siRNA) or overexpression and proliferation and transwell migration assays were performed to monitor the effect of manipulated survivin expression on the growth rate and migratory ability of the transfected cells. RESULTS: Among the HCC cell lines tested, we found high endogenous expression of survivin mRNA and protein in Mahlavu cells. After silencing survivin expression in Mahlavu cells, there was a dramatic decrease in the cell growth rate and an increase in the metastatic potential of the cells. Overexpression of survivin in Hep3B cells suppressed the ability of the cell to migrate. The mechanism of enhanced cell migration caused by decreased survivin expression is mediated through the downregulation of glucose-regulated protein 78 (GRP78) and the upregulation of the epithelial-mesenchymal transition (EMT) marker, vimentin. CONCLUSIONS: Survivin may mediate metastasis in HCC. The knockdown of survivin expression may enhance cancer metastasis through the downregulation of GRP78 and upregulation of vimentin expression.

Glucose-regulated protein 78 (GRP78) mediated the efficacy to curcumin treatment on hepatocellular carcinoma.

BACKGROUND: Glucose-regulated protein 78 (GRP78) plays an important role in the therapeutic treatment and progression of cancer. However, little is known about the effect of GRP78 expression to curcumin in hepatocellular carcinoma (HCC). MATERIALS AND METHODS: In this study, we generated GRP78 knockdown cells (GRP78KD) by a short interfering RNA (siRNA) technique. The antiproliferation effects of curcumin were determined by MTT assay, TUNEL assay, and cell cycle determination. RESULTS: We found that GRP78KD cells were more resistant to curcumin treatment compared with the parental cells in MTT assay. The apoptosis cell population was increased in scrambled-siRNA cells treated with curcumin compared with GRP78KD cells in cell cycle distribution and TUNEL assays. Finally, we found that knocking down GRP78 causes resistance to curcumin treatment through the suppression of caspase-3 and caspase-8 expression levels. CONCLUSIONS: We conclude that the expression level of GRP78 may contribute to the therapeutic effect of curcumin on HCC cells.

Normoxically overexpressed hypoxia inducible factor 1-alpha is involved in arsenic trioxide resistance acquisition in hepatocellular carcinoma.

BACKGROUND: The aim of this study was to examine the underlying signaling mechanisms of arsenic trioxide (ATO)-mediated anticancer effects and the responsible biomarker(s) for the acquired resistance in human heptatocellular carcinoma (HCC). MATERIALS AND METHODS: The therapeutic effects of ATO were examined using 2 characteristically distinct HCC cell lines, Hep-J5 (overexpressing HIF-1α/GRP78) and SK-Hep-1 (the matched control). ATO-mediated proliferation inhibition, oxidative stress, and apoptosis were analyzed using flowcytometric analysis and western blotting. The role of HIF-1α and GRP78 in HCC resistance to ATO treatment was determined using RNA silencing and inhibitor approaches. RESULTS: SK-Hep-1 cells, lacking both HIF-1α and GRP78 expressions were responsive to ATO-induced apoptosis via an oxidative-nitrosative mechanism. Intracellular glutathione depletion and lipid peroxidation have been identified as the early cascade of events preceding apoptosis via cytochrome c release and the severe drop of mitochondrial membrane potential (MMP). Conversely, Hep-J5 cells, with normoxic coexpression of HIF-1α and GRP78, were resistant to ATO-induced apoptosis. GRP78-silenced Hep-J5 cells remained resistant to ATO treatment. In contrast, ATO resistance in Hep-J5 cells was overcome by the addition of YC-1, a HIF-1α inhibitor. CONCLUSIONS: HIF-1α was identified as the major positive modifier for ATO resistance acquisition in HCC, and it represents a prime molecular target for overcoming ATO resistance.

Glucose-regulated protein 78 (GRP78) silencing enhances cell migration but does not influence cell proliferation in hepatocellular carcinoma.

BACKGROUND: GRP78 plays an essential role in embryonic development and in the therapeutic treatment and progression of cancer. However, little is known about the role of GRP78 in hepatocellular carcinoma (HCC). METHODS: In this study, we characterized five different HCC cell lines to examine GRP78 expression patterns and found that only HepJ5 cells ectopically overexpress GRP78. We knocked down GRP78 expression in HepJ5 cells using a small interfering RNA (siRNA), and the proliferation assay and migration assay were performed. RESULTS: Using siRNA technique, we could successfully reduce GRP78 expression levels in HepJ5 cells. In a cell growth study, we found that GRP78-siRNA caused no significant changes in cellular proliferation in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), colony formation, and cell cycle distribution. In a cell migration study, we found that GRP78-siRNA HepJ5 cells had dramatically increased migration ability in Transwell assay. CONCLUSIONS: We conclude that ectopically expressed GRP78 does not contribute to the increased proliferation of HepJ5 cells, but does correlate with the migration of HCC cells under normoxic conditions.

Glucose-regulated protein 78 is a novel contributor to acquisition of resistance to sorafenib in hepatocellular carcinoma.

BACKGROUND: Sorafenib is a newly established cancer drug found to be an effective systemic treatment for advanced hepatocellular carcinoma (HCC). However, little is known about any potential effectors that modify tumor cell sensitivity towards sorafenib. Here, we present the first evidence that glucose-regulated protein 78 (GRP78) is intimately associated with acquisition of resistance towards sorafenib. METHODS: The role of GRP78 in acquisition of resistance towards sorafenib was determined using HepJ5 (a GRP78-overexpressing subline) and HepG2 as its pair-matched control. RNA interference in cancer cells was applied to determine the influence of GRP78 expression on sensitivity to sorafenib treatment. RESULTS: We found that HepG2 cells exhibited higher sensitivity toward sorafenib, with 50% inhibition concentration (IC(50)) >20 microMu for HepJ5 and 4.8 microM for HepG2. Specifically, when HepG2 cells received 20 microM sorafenib treatment for 24 h, over 80% of cells underwent apoptosis compared with only 32% of HepJ5 cells under similar experimental conditions. Similarly, GRP78 knockdown in HepJ5 cells by small interfering RNA (siRNA) technique enhanced the efficacy of sorafenib-mediated cell death. This was reflected by a shift of IC(50) values from >20 microM to 4.8 microM. CONCLUSIONS: GRP78 is a positive modifier for sorafenib resistance acquisition in HCC and represents a prime target for overcoming sorafenib resistance.

Is the blood donated by habitual nut quid chewers suitable for use in transfusion?

BACKGROUND/PURPOSE: Betel quid (BQ) chewing is a popular oral masticatory activity, and there are approximately 600 million BQ chewers worldwide. Although chewing BQ has been linked to the patho-genesis of oral cancer, leukoplakia, and oral submucous fibrosis. The question whether the mixed constituents present in areca nut, which may exert cytotoxic effects on red blood cells (RBCs), has never been addressed. METHODS: Heparinized blood specimens were obtained with informed consent from healthy laboratory personnel. RBCs were separated with the standard procedure and adjusted to 10% hematocrit with PBS. Various concentrations of areca nut extract (ANE; 100-800 microg/mL) were added to these RBC preparations and incubated at 37 degrees C for 4 hours. Two portions (0.4 mL each) of the incubated RBCs were then used for measuring osmotic deformability index and for observing RBC morphology with scanning electron microscopy. The remaining RBCs were used for determining membrane sulfhydryl groups and protein profiles by sodium dodecyl sulfate polyacrylamide gel electrophoresis. RESULTS: Blood incubated with various concentrations of ANE showed concentration-dependent decreases in osmotic deformability index and membrane sulfhydryl groups. Membrane protein profiles revealed a significant loss of the band 3 fraction, with the concomitant appearance of several new protein bands in the electropheretogram. Finally, drastic morphological changes of ANE-treated RBCs were observed. CONCLUSION: We suggest that to assure the quality of transfusion, the blood donated by a habitual BQ chewer should be used with caution because of its possible contamination with areca nut ingredients that may be cytotoxic to RBCs.

Cholestin (Monascus purpureus rice) inhibits homocysteine-induced reactive oxygen species generation, nuclear factor-kappaB activation, and vascular cell adhesion molecule-1 expression in human aortic endothelial cells.

Hyperhomocysteinemia is associated with dysfunction and an independent risk factor of cardiovascular diseases. Cholestin (Monascus purpureus-fermented rice), contains a naturally-occurring statin, which has lipid-modulating and anti-inflammatory effects. We investigated the effects of Cholestin extract on the expression of vascular cell adhesion molecule-1 (VCAM-1) by homocysteine (HCY)-treated human aortic endothelial cells (HAECs). Supplement of HAECs with Cholestin extract significantly suppressed cellular binding between the human monocytic cells U937 and HCY-stimulated HAECs. Quantitative PCR and immunoblot analysis showed that Cholestin extract significantly attenuated HCY-induced expression of VCAM-1 mRNA and protein, respectively. Gel shift assays showed that Cholestin treatment reduced HCY-activated transcription factor nuclear factor-kappaB (NF-kappaB). Furthermore, Cholestin also attenuated reactive oxygen species (ROS) generation in vitro and in HCY-treated HAECs. Supplement with statins including simvastatin and parastatin gave similar results as compared with Cholestin. In conclusion, Cholestin reduces HCY-stimulated endothelial adhesiveness as well as downregulating intracellular ROS formation, NF-kappaB activation, and VCAM-1 expression in HAECs, supporting the notion that the natural compound Cholestin may have potential implications in clinical atherosclerosis disease.

Isoobtusilactone A induces both caspase-dependent and -independent apoptosis in Hep G2 cells.

Isoobtusilactone A, a constituent isolated from the leaves of Cinnamomum kotoense, has been demonstrated by us earlier to be an agent capable of inducing apoptotic cell death of Hep G2 cells. In order to clarify if caspases alone were the sole mediator for eliciting this apoptotic process, a broad caspases inhibitor, Z-VAD.fmk, was utilized to explore this possibility. Interestingly, although Z-VAD.fmk was demonstrated to be capable of completely inhibiting isoobtusilactone A-induced oligonucleosomal DNA fragmentation, yet it could only prevent limited amount of cells from becoming apoptosis-prone. These data implied that some other mechanism(s) might be involved. Thus, the involvement of apoptosis-inducing factor (AIF), a mediator arbitrating caspase-independent apoptosis, in isoobtusilactone A-induced apoptotic process was examined. These findings indicated that isoobtusilactone A could elicit the nuclear translocation of AIF that accompanied the occurrence of large-scale DNA fragmentation. Reduction of AIF expression by AIF-siRNA transfection suppressed large-scale DNA fragmentation. Interestingly, inhibition of AIF expression by AIF-siRNA could not prevent isoobtusilactone A-induced oligonucleosomal DNA fragmentation. In the same vein, when the cells were simultaneously combined pretreatment with AIF-siRNA and Z-VAD.fmk, both large-scale DNA and oligonucleosomal DNA fragmentations could nearly be prevented. Taken together, these findings suggested that isoobtusilactone A-induced apoptotic cell death was mediated via both caspase-dependent and -independent pathways.

(-)-Anonaine induces apoptosis through Bax- and caspase-dependent pathways in human cervical cancer (HeLa) cells.

(-)-Anonaine has been shown to have some anticancer activities, but the mechanisms of (-)-anonaine inducing cell death of human cancer cells is not fully understood. We investigated the mechanisms of apoptosis induced by (-)-anonaine in human HeLa cancer cells. Treatment with (-)-anonaine induces dose-dependent DNA damage that is correlated with increased intracellular nitric oxide, reactive oxygen species, glutathione depletion, disruptive mitochondrial transmembrane potential, activation of caspase 3, 7, 8, and 9, and poly ADP ribose polymerase cleavage. Our data indicate that (-)-anonaine up-regulated the expression of Bax and p53 proteins in HeLa cancer cells. The apoptosis and expression of Bax induced by (-)-anonaine could be inhibited when the HeLa cells were pretreated with Boc-Asp(OMe)-fmk, which is a broad caspases inhibitor. There was no obvious DNA damage in the (-)-anonaine-treated Madin-Darby canine kidney and Vero cell lines. Both Madin-Darby canine kidney and Vero cell lines are kidney epithelial cellular morphology. These results suggest that (-)-anonaine might be considered a potent compound for chemotherapy against cervical cancer or a health food supplement for cancer chemoprevention.

Distinct differences in the induction of stimulus-mediated superoxide generation by polymorphonuclear neutrophils isolated from patients with different types of leukemia.

BACKGROUND/PURPOSE: Accumulating literature has documented that there exists a distinct difference in nitro blue tetrazolium reduction capacity by the polymorphonuclear neutrophils (PMNs) from patients with different types of leukemia. The underlying mechanism associated with this observed phenomenon remains to be clarified. METHODS: The production of O2-, monitored by a validated probe (lucigenin)-based ultraweak chemiluminescence, in resting and/or phorbol-1,2-myristate-1,3-acetate (PMA)- and zymosan-stimulated systems of various leukemic PMNs was measured. In parallel with these studies, we also quantified superoxide dismutase isozymes (Cu, Zn-SOD, Mn-SOD) from these isolated PMNs by established methods. RESULTS: A marked increase was observed in O2- generation by the PMNs from patients with acute myeloid leukemia (AML), and chronic myeloid leukemia (CML), but not from patients with acute lymphocytic leukemia (ALL) when compared with controls either in the resting condition or after being stimulated by either PMA or zymosan. In parallel, we also quantified SOD isozyme activities and found that the total and CuZn-SOD of PMNs from AML were indeed significantly lower than either controls or ALL, implying that higher levels of O2- generation might result from a deficiency in this O2- metabolizing enzyme. CONCLUSION: Our data suggest that a distinct difference in the capability of O2- generation under stimulated conditions between PMNs from ALL and AML (or CML) may be of potential taxonomic or even therapeutic usefulness.

γ-Glutamylcysteine synthetase (γ-GCS) as a target for overcoming chemo- and radio-resistance of human hepatocellular carcinoma cells.

AIMS: This study uncovered that the genetically endowed intracellular glutathione contents (iGSH) regulated by the catalytic subunit of γ­glutamylcysteine synthetase heavy chain (γ­GCSh) as a prime target for overcoming both the inherited and stimuli-activated chemo- and radio-resistance of hepatocellular carcinoma (HCC) cells. MAIN METHODS: Reactive oxygen species (ROS) production and mitochondrial membrane potential (Δψm) were determined by the probe-based flow cytometry. The TUNEL assay was used as an index of radio-sensitivity and the MTT assay was used as an index of chemo-sensitivity against various anti-cancer agents. iGSH and γ­GCSh activity were measured by HPLC methods. γ­GCSh-overexpressing GCS30 cell line was established by tetracycline-controlled Tet-OFF gene expression system in SK-Hep-1 cells. KEY FINDINGS: The relative radio-sensitivities of a panel of five HCC cells were found to be correlated negatively with both the contents of iGSH and their corresponding γ­GCSh activities with an order of abundance being Hep G2 > Hep 3B > J5 > Mahlavu > SK-Hep-1, respectively. Similarly, the cytotoxicity response patterns of these HCC cells against arsenic trioxide (ATO), a ROS-producing anti-cancer drug, were exactly identical to the order of ranking instigated by the radiotherapy (RT) treatment. Next, γ­GCSh-overexpressing GCS30 cells were found to possess excellent ability to profoundly mitigate both the drop of Δψm and apoptotic TUNEL-positive cell population engendered by ATO, cisplatin, doxorubicin, and RT treatments. SIGNIFICANCE: Our data unequivocally demonstrate that γ­GCSh may represent a prime target for overcoming anti-cancer drugs and RT resistance for HCC cells.

A novel enzymatic method for determination of homocysteine using electrochemical hydrogen sulfide sensor.

Homocysteine is a sulfur-containing compound produced during metabolism process of methionine. Its uptake in human plasma is believed to be the cause of cardiovascular diseases and many other diseases. An electrochemical method was proposed for selective and quantitative measurement of homocysteine by employing hydrogen sulfide sensor coupled with methionine a, g-lyase. The principle of this method is to measure the evolved hydrogen sulfide from the enzymatic reaction between homocysteine and methionine a, g-lyase. The sensitivities of the measurements at different pH values of the tris buffer solutions and at room temperature peaked to 275 pA/mM at pH 6.5 with detection limit of 150 nM (based on 3 s cutoff). The linearity measurements at pH 6.5 were performed for the homocysteine concentrations range from 0.5 to 200 mM, which is wider than the human blood plasma total homocysteine level of 5 to 100 mM, and the regressive analysis of the experiments gave R2=0.9987. The enzyme also showed the fastest response to homocysteine in the tris buffer solution of pH 7.5 with the current approaching its maximum at 134 seconds. The interference tests against several common agents were carried out, and found that cysteine and methionine were the major two species to introduce measurement problem. The solution to this interference problem was explored and discussed thoroughly based on the preliminary tests. The sensitivities of the experiments against several enzyme concentrations were also performed.

Antioxidant status and superoxide anion radical generation in acute myeloid leukemia.

OBJECTIVES: This study was undertaken to investigate if there is a disparity in the antioxidant status and the ability of superoxide anion (O(2)(-)) generation in the patients with acute myeloid leukemia (AML). DESIGN AND METHODS: The peripheral blood samples from thirty AML patients and thirty-six healthy subjects were collected and leukocytes, erythrocytes and plasma were separated for use in various parameter measurements. RESULTS: The generation of O(2)(-), as reflected by lucigenin-based CL (LBCL), by the leukocytes of patients with AML was found to be significantly elevated either in resting or stimuli-elicited condition as compared with that of healthy controls (p<0.05). Coincidentally, these data were matched up with the suppressed SOD activities, notably in Cu/Zn SOD isoform found in AML patients (p<0.05). Conversely, SOD and GPx activities in erythrocytes of patients with AML were shown to be significantly higher than their normal counterparts (p<0.05). CONCLUSIONS: These data suggest that altered expression of antioxidant enzymes and higher capability of O(2)(-) generation by leukocytes seem to be a distinct feature of AML.

The efficacy of protective effects of tannic acid, gallic acid, ellagic acid, and propyl gallate against hydrogen peroxide-induced oxidative stress and DNA damages in IMR-90 cells.

There is increasing evidence that reactive oxygen species (ROS) are intimately involved in the oxidative damage of tissues for a wide variety of pulmonary diseases. Thus, it is desirable to search for chemopreventive agents that can counteract ROS-mediated injury to the pulmonary tissues. Using a human lung fibroblast IMR-90 cells as the experimental model, we first demonstrated that nearly 90% of intracellular ROS could be removed when H(2)O(2)-treated cells (200 microM) simultaneously incubated with 10 microg/mL of tannic acid (TA), gallic acid (GA), ellagic acid (EA), and propyl gallate (PA). Using C(11)-BODIPY(581/591 )as a lipid peroxidation probe, we also attested that all these compounds examined (10 microg/mL) could alleviate H(2)O(2)-evoked lipid peroxidation phenomena. Next, we examined the protective effects of these compounds on the depletion of intracellular glutathione (iGSH) in H(2)O(2)-treated cells using CMF-DA probe. Interestingly, PA was demonstrated to be the only compound that could effectively protect the integrity of iGSH from being depleted by this system. Finally, the protective effects of these compounds against oxidative DNA damage were evaluated using 8-oxoguanine formation as a marker. Our data indicated that all four compounds suppressed the formation of 8-oxoguanine effectively. Taken together, our data suggested that TA, GA, EA, and PA can protect cells from oxidative stress.

6-shogaol (alkanone from ginger) induces apoptotic cell death of human hepatoma p53 mutant Mahlavu subline via an oxidative stress-mediated caspase-dependent mechanism.

Mahlavu cells, poorly differentiated and p53 mutants of a human hepatoma subline, are known to be highly refractory to a number of chemotherapeutic agents and radiotherapy due to their high expressions of multidrug resistance gene-1 (MDR-1) and Bcl-2 proteins. Thus, it is desirable to search for an alternative strategy for effective eradication of this type of cancer cells. We present evidence here for the first time that 6-shogaol (6-SG), an alkanone isolated from the rhizomes of ginger, can effectively induce apoptotic cell death of Mahlavu cells via an oxidative stress-mediated caspase-dependent mechanism. The cascade of events in 6-SG-induced apoptosis of these cells involved an initial overproduction of reactive oxygen species (ROS) followed by a severe depletion of intracellular glutathione (GSH) contents. Both events consequently entailed a significant drop in mitochondrial transmembrane potential (DeltaPsim), which ultimately activated the activities of caspases 3/7 resulting in the DNA fragmentation. Interestingly, we also found that N-acetylcysteine (NAC), an antioxidant and a precursor of GSH biosynthesis, could offer a near complete protection of apoptotic cell death exerted by 6-SG. Similarly, exogenously added GSH could also provide protection with an equal efficacy. However, it was paradoxical that both Boc-Asp(OMe)-fmk (a broad caspases inhibitor) and cyclosporin A (an mitochondrial permeability transition opening inhibitor) could only partially protect these cells from 6-SG-induced apoptosis. Taking these data into consideration, it is obvious that GSH depletion is the major contributing factor in arbitrating 6-SG-induced apoptosis of Mahlavu cells. In conclusion, we provide here a novel modality that can help to eradicate a p53 mutant of human hepatoma cells by using a natural consistent isolated form of ginger. These data also provide evidence to reaffirm the notion that consumption of certain foodstuffs can be beneficial to health because some of the constituents contained in them may be anticarcinogenic.

Isoobtusilactone A-induced apoptosis in human hepatoma Hep G2 cells is mediated via increased NADPH oxidase-derived reactive oxygen species (ROS) production and the mitochondria-associated apoptotic mechanisms.

Chemoprevention by the use of naturally occurring substances is becoming a promising strategy to prevent cancer. In this study, the effects of isoobtusilactone A, a novel constituent isolated from the leaves of Cinnamomum kotoense, on the proliferation of human hepatoma Hep G2 cells were studied. Under our experimental conditions, isoobtusilactone A was found to elicit a concentration-dependent growth impediment (IC(50)=37.5 microM). The demise of these cells induced by isoobtusilactone A was apoptotic in nature, exhibiting a concentration-dependent increase in sub-G(1) fraction and DNA fragmentation. Subcellular fractionation analysis further revealed that Bax translocation to mitochondria resulted in a rapid release of cytochrome c, followed by activation of caspase 3 and PARP cleavage, and finally cell death. Isoobtusilactone A-treated cells also displayed transient increase of ROS during the earlier stage of the experiment, followed by the disruption of mitochondrial transmembrane potential (DeltaPsi(m)). The presence of a ROS scavenger (N-acetyl-L-cysteine) and an inhibitor of NADPH oxidase (diphenyleneiodonium chloride) blocked ROS production and the subsequent apoptotic cell death. In addition, in order to investigate the acute toxicity of isoobtusilactone A, groups of 5-6-week old Sprague-Dawley rats were subjected to oral administration of 350, or 700 mg/kg bw isoobtusilactone A four times each week for two weeks. There was no significant difference between control animals and treated animals with respect to the body weight gain, the body weight ratio of liver, spleen and kidney, haematological and clinical chemistry parameters. Taken together, our data suggest that ROS generated through the activation of NADPH oxidase plays an essential role in apoptosis induced by isoobtusilactone A, and the dosages of isoobtusilactone A tested in this study did not cause animal toxicity.

Protective effects of a freeze-dried extract of vegetables and fruits on the hydroxyl radical-mediated oxidative damage of DNA and decrease of erythrocytes deformability.

The protective effects of a freeze-dried extracts of vegetables and fruits (BauYuan; BY) on the hydroxyl radical-mediated DNA strand breakages and the structural integrity of human red blood cells (RBCs) were investigated. First, the supercoiled plasmid (pEGFP-C1) DNA was subjected to oxidation damage by an ascorbate-fortified Fenton reaction and the protective effects were analyzed by agarose gel electrophoresis. In the absence of BY extracts, exposure of the high-throughput .OH-generating system (Fe2+ concentration >1.0 microM) caused a complete fragmentation of DNA. Supplementation of BY extract (1 mg/mL) to the plasmid DNA prior to the exposure could prevent it significantly. In contrast, as the plasmid exposed to a low-grade .OH-generating system (Fe2+<0.1 microM), the BY extract (1 mg/mL) provided an almost complete protection. Next, the cell deformabilities were measured to assess the protection effects of various BY extracts on human erythrocytes exposed to the oxidative insults. We found that both the aqueous extract and the organic solvent-derived extracts could strongly protect human RBCs from the reactive oxygen species (ROS)-mediated decrease in the deformability indices. The results implicated that the BY extracts could effectively protect the cell membrane integrity via scavenging ROS which enabling RBCs to maintain a balance of water content and surface area to prevent the drop of cell deformability.

Genetic and cellular characterizations of human TCF4 with microsatellite instability in colon cancer and leukemia cell lines.

It has been reported that the mutational inactivation of the adenomatous polyposis coli (APC) and beta-catenin genes play important roles in colorectal carcinogenesis. However, alteration of the components in the Wnt signaling pathway in colorectal cancer (CRC) with microsatellite instability (MSI) has been elucidated. To define the precise role of the Wnt signaling components in CRC and leukemia cell lines with MSI, mutational analyses of the T cell factor 4 (TCF4) genes were performed. Here we describe for the first time a TCF4 MSI+ phenotype in leukemia cell lines except in colon cancer cell lines. Moreover, we found that these cell lines exhibited deletion and insertion of 1-2A in an (A)9 repeat so as to result in (A)7, (A)8, (A)10 and (A)11 repeat, respectively. To characterize the cellular function of these special TCF4 mutant clones, transient transfection and fluorescent microscopy were analyzed and the results revealed that the TCF4 frameshift gene products all localized in nuclei. Surprisingly, these TCF4 frameshift mutants lost transcriptional activity with beta-catenin and down-regulate the target gene expression. These results delineate a novel role for MSI+TCF4 in leukemia and colon cancer progression.

Development of probe-based ultraweak chemiluminescence technique for the detection of a panel of four oxygen-derived free radicals and their applications in the assessment of radical-scavenging abilities of extracts and purified compounds from food and herbal preparations.

We report here the development of a probe-based ultraweak chemiluminescence (uwCL) method capable of detecting a panel of four oxygen-derived free radicals (ODFRs) including superoxide (O2-), hydrogen peroxide (H2O2), hydroxyl radical (*OH), and peroxyl radical (ROO*) using different probes specific for these radicals performed by the same uwCL analyzer. The selected radical-generating systems and their corresponding uwCL-probing emitters were validated. These ODFR-detecting systems were subsequently utilized by us to assess the radical-scavenging ability (RSA) of a variety of extracts and purified constituents derived from foods and herbal preparations. Our approach for assessing RSA for these constituents is based on the suppression of uwCL generated by each ODFR, and the degrees of inhibition have been shown to be dose-dependent. For this reason, the estimation of IC50 for each testing compound can be obtained from the curve constructed based on the percent of inhibitions of uwCL versus the concentrations of the compound tested. To illustrate the practical applications of our devised methodology, data for comparative studies of RSA activities of fermented extracts of Cordeceps sinensis, purified methylgallate isolated from Toona sinesis, resveratrol purified from grape seeds, plus epimedin C from the aerial part of the Epimedium plant (yinyanghuo) are to be presented.