Biomedical subjects

S Y Lin-Shiau

Publications and source records attributed to S Y Lin-Shiau.

At least 19 recordsLinked to original sources

PubMed2000

Activation of transcription factors of nuclear factor kappa B, activator protein-1 and octamer factors in hyperalgesia.

Involvement of c-fos and neuronal nitric oxide synthase (nNOS) in the hyperalgesia induced by complete Freund adjuvant (CFA) has been reported. In this paper, we attempted to investigate whether the transcription factors regulating the gene expression of c-fos and nNOS, including activator protein-1 (AP-1), nuclear factor kappa B (NF-kappa B), and octamer factors (Oct), are activated by CFA during the development of hyperalgesia. The electrophoretic mobility shift assay (EMSA) was used to determine whether there were changes in the transcription factors in the lumbar spinal cord of adult rats following subcutaneous injection of CFA in one hindpaw of the rats. Maximum binding of AP-1, NF-kappa B and Oct was found at 0.5, 1 and 2 h after CFA injection, respectively. These findings suggest that the activation of these transcription factors is pivotal for the expression of c-Fos and nNOS proteins, which reached a peak at 3 and 48 h after CFA injection, respectively. The behavioral testing of hyperalgesia demonstrated that CFA reduced the thresholds for mechanical and thermal algesia, reaching a minimum at 6 h. The thresholds had only partially recovered after 96 h. Based on these findings, we conclude that AP-1, NF-kappa B and Oct are crucial for the expression of c-Fos proteins at an early stage (at 3 h) and for the expression of nNOS at a late stage of hyperalgesia (48 h post-injection) induced by CFA.

Animals↗

PubMed2000

Suramin protects the murine motor nerves from the toxic effects of presynaptic Ca(2+) channel inhibitors.

The purpose of this study is to investigate whether suramin is capable of preventing the neurotoxic effects of Ca(2+) channel inhibitors at the presynaptic sites. Mouse diaphragm and triangularis sterni preparations were used for this study in order to measure the muscle tension and nerve terminal Ca(2+) current, respectively. Both omega-conotoxin MVIIC and omega-agatoxin IVA markedly inhibit the nerve-evoked muscle contractions as well as the nerve terminal Ca(2+) current respectively. Pretreatment with suramin (0.3 mM) significantly reduced the inhibitory effect of nerve-evoked muscle contractions and Ca(2+) current induced by either omega-conotoxin MVIIC or omega-agatoxin IVA but not that induced by the non-selective Ca(2+) channel blocker, Cd(2+). Neither suramin nor Ca(2+)-channel toxins significantly affect Na(+)- and K(+) currents of the nerve terminals. These findings indicate that suramin selectively interferes the action of presynaptic Ca(2+) channel neurotoxins and thus reduces their depressant effects on the muscle contractions. The implication of these findings is that suramin and its derivatives may potentially become useful agents in management of intoxication of Ca(2+) channel neurotoxins.

Animals↗

PubMed2000

High glucose-induced apoptosis in human endothelial cells is mediated by sequential activations of c-Jun NH(2)-terminal kinase and caspase-3.

BACKGROUND: Diabetes mellitus causes multiple cardiovascular complications. High glucose can induce reactive oxygen species and apoptosis in endothelial cells. Little is known about the molecular mechanisms in high glucose-induced endothelial cell apoptosis. METHODS AND RESULTS: We elucidated the signaling pathway of high glucose-induced apoptosis in human umbilical vein endothelial cells (HUVECs). HUVECs were treated with media containing 5.5, 19, or 33 mmol/L of glucose in the presence or absence of an antioxidant, ascorbic acid. The level of intracellular H(2)O(2) was measured by flow cytometry. For detection of apoptosis, the cell death detection ELISA assay and the morphological Hoechst staining were used. High glucose was capable of inducing the activity of c-Jun NH(2)-terminal kinase (JNK) but not extracellular signal-regulated kinase 1/2 or p38 mitogen-activated protein kinase during the treatment periods, as evidenced by immunocomplex kinase assay. Moreover, we found that the interleukin 1beta-converting enzyme (ICE)/CED-3 family protease (caspase-3) became activated in high glucose-induced apoptosis. Caspase-3/CPP32-specific inhibitor, Ac-DEVD-CHO, could inhibit high glucose-induced apoptosis. Furthermore, we found that JNK1 specific antisense oligonucleotide could suppress caspase-3 activity but not affect H(2)O(2) generation and could block apoptosis induced by high glucose. Also, H(2)O(2) generation, JNK activity, caspase-3 activity, and the subsequent apoptosis induced by high glucose could be suppressed by ascorbic acid. CONCLUSIONS: The present study indicates that reactive oxygen species induced by high glucose may be involved in JNK activation, which in turn triggers the caspase-3 that facilitates the apoptosis in HUVECs.

Antisense Elements (Genetics)↗

PubMed2000

The effects of inhaled nitric oxide, gabexate mesilate, and retrograde flush in the lung graft from non-heart beating minipig donors.

BACKGROUND: The use of lung grafts from non-heart-beating donors (NHBD) is one way of solving the donor organ shortage problem. In this experiment, we studied the effect of retrograde flush (RF) from the left atrium before harvest, inhaled nitric oxide (NO), and gabexate mesilate (FOY), a protease inhibitor, in the lung grafts from NHBD. METHODS: Forty-eight Lee-Sung, small-ear, miniature pigs (15-20 kg) were divided into 24 pairs (donor and recipient) and four groups. The donor lungs were flushed and harvested 90 min after cardiac arrest. No i.v. heparin was administered until the time before flush and harvest. Left single lung transplantation was undertaken, and the recipients were observed for 18 hr. The grafts warm and cold ischemia times were 90 (controlled) and 183+/-23.4 min. Group 1 (untreated control, UC, n=6) had core perfusion through a Swan-Ganz catheter followed by a single, antegrade flush with modified Euro-Collin's solution containing heparin, urokinase, and PGE1. Group 2 (RF group, n=6) had the same as group 1, except that one additive retrograde flush through the left atrium was administered. Group 3 (NO group, n=6) had the same as group 1, except that 20 parts per million (ppm) inhaled NO was administered for the cadaver donors before the graft harvest, and for the recipients after the grafts reperfusion. Group 4 (FOY group, n=6) had the same as group 1, except that the recipients received FOY i.v. infusion from the beginning of the recipient's operation and continuously throughout the experiments. RESULTS: Compared with the group 1 (control), group 2 (RF) had significantly (P<0.05) lower mean pulmonary artery pressure, pulmonary vascular resistance (PVR), lung wet/dry ratio, histological lung injury score, and higher PaO2/FiO2 and pulmonary dynamic compliance. Group 3 (NO) had significantly lower mean pulmonary arterial pressure, PVR, lung injury score, degree of tissue neutrophils infiltration (histological and myeloperoxidase assay), bronchoalveolar lavage fluid protein content and neutrophils (PMNs) percentage, and higher PaO2/FiO2 and pulmonary dynamic compliance. Group 4 (FOY) had significantly lower PMNs infiltration, lung injury score, wet/dry ratio, bronchoalveolar lavage fluid protein and PMNs percentage, and higher PaO2/FiO2. Group 2 (RF) revealed better gas exchange (PaO2/FiO2) than the control (group 1) at earlier reperfusion periods (1st and 5th hr). On the contrary, group 4 (FOY) had higher PaO2/FiO2 than group 1 only at later period (18th hr). Pathologically, retrograde flush (group 2, RF) inhibited the intravascular thrombi formation more effectively than the NO or FOY treatment. However, the NO or FOY treatment inhibited the neutrophil infiltration more effectively than did the retrograde flush. CONCLUSION: The retrograde flush, inhaled NO and FOY infusion are beneficial to the protection of the NHBD lung grafts at an early reperfusion period, through different mechanisms. The use of these treatments in combination might help us to find a better way to protect the NHBD grafts against the preservation and reperfusion injury.

Administration, Inhalation↗

PubMed2000

Suppression of lipopolysaccharide-induced nuclear factor-kappaB activity by theaflavin-3,3'-digallate from black tea and other polyphenols through down-regulation of IkappaB kinase activity in macrophages.

We investigated the inhibition of IkappaB kinase (IKK) activity in lipopolysaccharide (LPS)-activated murine macrophages (RAW 264.7 cell line) by various polyphenols including (-)-epigallocatechin-3-gallate, theaflavin, a mixture of theaflavin-3 gallate and theaflavin-3'-gallate, theaflavin-3,3'-digallate (TF-3), pyrocyanidin B-3, casuarinin, geraniin, and penta-O-galloyl-beta-D-glucose (5GG). TF-3 inhibited IKK activity in activated macrophages more strongly than did the other polyphenols. TF-3 strongly inhibited both IKK1 and IKK2 activity and prevented the degradation of IkappaBalpha and IkappaBbeta in activated macrophage cells. The results suggested that the inhibition of IKK activity by TF-3 could occur by a direct effect on IKKs or on upstream events in the signal transduction pathway. Furthermore, geraniin, 5GG, and TF-3 all blocked phosphorylation of IKB from the cytosolic fraction, inhibited nuclear factor-kappaB (NFkappaB) activity, and inhibited increases in inducible nitric oxide synthase levels in activated macrophages. These results suggest that TF-3 may exert its anti-inflammatory and cancer chemopreventive actions by suppressing the activation of NFkappaB through inhibition of IKK activity.

Animals↗

PubMed2000

Death signaling pathway induced by pyrrolidine dithiocarbamate-Cu(2+) complex in the cultured rat cortical astrocytes.

The chelating and antioxidant effects of pyrrolidine dithiocarbamate (PDTC) have been investigated extensively for preventing cell death induced by different insults. However, the toxic effects of PDTC have been studied only recently and fewer studies on the toxic effects on astrocytes have been reported. In our study, we demonstrated that both PDTC and Cu(2+) alone were rated as only weakly toxic in inducing cell death in cortical astrocytes with IC(50) of 300 microM and 180 microM, respectively. However, PDTC and Cu(2+) in the complex form markedly potentiated with each other by about 1,000-fold with IC(50) of 0.3 microM PDTC plus 10 microM Cu(2+). Other metals at concentrations of 3-10 microM (VO(4)(5+), Cr(6+), Mn(2+), Fe(2+), Co(2+), Ni(2+), Zn(2+), Pb(2+), Bi(2+), Ba(2+), UO(2+), Cs(+), SeO(4)(2-), La(3+)) had no such potentiating effects on PDTC. Changes in morphology (nuclear condensation), apoptotic body formation, and hypodiploidity of DNA suggested that the PDTC-Cu(2+) complex induced cell death through an apoptotic process. Further studies showed that the PDTC-Cu(2+) complex decreased mitochondrial membrane potential, increased hydrogen peroxide production, and depleted GSH contents. After the increased oxidative stress, PDTC-Cu(2+) complex differentially activated JNKs, ERK, p38 and caspase 3, which caused PARP degradation in a time-dependent manner. All these effects were consistent with the increased cellular Cu contents. The nonpermeable copper-specific chelator bathocuproine disulfonate (BCPS), but not the permeable Cu(2+) chelator neocuproine, abolished all the observed effects. Antioxidants (N-acetylcysteine [NAC], vitamin C), catalase, and Cu(2+)-binding proteins (albumin, hemoglobin, and higher serum) reduced the cytotoxic effects of PDTC-Cu(2+) complex. We concluded that the death signaling pathway of PDTC-Cu(2+) complex was mediated by oxidative stress and subsequent JNK activation. These findings imply that PDTC, a widely used pesticide and medicine that is capable of penetrating the blood-brain barrier, may cause neurotoxicity through astrocyte dysfunction.

Animals↗

PubMed2000

Alterations in the properties and isoforms of sciatic nerve Na(+), K(+)-ATPase in methylcyclopentadienyl manganese tricarbonyl-treated mice.

The in vivo effect of methylcyclopentadienyl manganese tricarbonyl (MMT), an organic manganese-containing compound, on the mouse motor nerve was studied. The motor nerve conduction velocity was markedly decreased in MMT-treated mice. The Na(+),K(+)-ATPase activity of sciatic nerve isolated from MMT-treated mice was decreased; however, the sciatic nerve Na(+),K(+)-ATPase activity was not affected by the in vitro treatment of MMT. Moreover, [(3)H]ouabain binding of sciatic nerve isolated from MMT-treated mice was decreased. Using Western blot analysis, the amount of Na(+),K(+)-ATPase catalytic alpha1 subunit polypeptide in sciatic nerve of MMT-treated mice was also decreased. These results indicate that a causal relationship may exist between reduced nerve Na(+),K(+)-ATPase activity and motor nerve conduction velocity in MMT-treated mice and that a measurable decrease in alpha1 catalytic subunit isoform of Na(+),K(+)-ATPase may be necessary for the development of peripheral neuropathy by MMT.

Animals↗

PubMed2000

Activities of Na(+),K(+)-ATPase and Ca(2+)-ATPase in cochlear lateral wall after acoustic trauma.

Na(+),K(+)-ATPase and Ca(2+)-ATPase are well known participants in the active transport of ions in the inner ear. These two enzymes play an important role in maintaining cochlear function. Although changes in these enzymes' activities in the cochlea have been implicated in noise-induced hearing loss, no evidence of quantitative alteration of Na(+),K(+)-ATPase or Ca(2+)-ATPase activities has ever been shown. The present study was undertaken to determine the quantitative alterations of their activities by microcolorimetric assay in the cochlear lateral wall after acoustic trauma. Adult albino guinea pigs were exposed to white noise at 105+/-2 dB A for 10 min or 40 h. The age-matched control animals were not exposed to noise. Noise exposure resulted in a significant threshold shift of the auditory brainstem response (P<0.001). Significant decreases in activities of Na(+),K(+)-ATPase and Ca(2+)-ATPase were found in the cochlear lateral wall after noise exposure (P<0.001). Statistical analysis indicated that a good correlation held not only between the decline of these enzyme activities and noise-induced hearing loss, but also between the gradual partial recovery of these parameters during the first 10-day recovery period. The present findings suggest that metabolic damage and ionic disturbance may contribute, at least partially, to noise-induced hearing threshold shift.

Animals↗

PubMed2000

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.

Antioxidants↗

PubMed2000

Induction of apoptosis by lovastatin through activation of caspase-3 and DNase II in leukaemia HL-60 cells.

Lovastatin, an HMG-CoA reductase inhibitor, was found to suppress growth and induce apoptosis in culture human promyelocytic leukaemic cell, HL-60. However, the mechanisms of lovastatin-induced apoptosis are still unclear. In this study, we attempted to elucidate the signal transduction pathway for lovastatin-induced apoptosis in HL-60 cells in a dose- and time-dependent manner. The features of this apoptosis were attenuated by the presence of mevalonate, a metabolic intermediate of cholesterol synthesis. Treatment of lovastatin caused a rapid release of mitochondrial cytochrome c into cytosol and subsequent induction of caspase-3, but not caspase-1 activity. Lovastatin also stimulated proteolytic cleavage of poly-(ADP-ribose) polymerase (PARP), and followed by the appearance of caspase activity and DNA fragmentation. Pretreatment with caspase-3 inhibitors, Ac-DEVD-CHO and Z-VAD-FMK, inhibited lovastatin induced caspase-3 activity and DNA fragmentation. Furthermore, we demonstrated that DNase II was involved in the DNA fragmentation induced by lovastatin. These results suggested that the mechanism of lovastatin induced HL-60 cells apoptosis through activation of caspase-3 and DNase II activities.

Apoptosis↗

PubMed2000

Astrocytes modulate thapsigargin-induced changes in calcium concentration and neuronal survival.

When mature cerebellar granule neurons (CGN) grown in high K+ (25 mM K+, HK)-serum containing medium are subjected to the HK/serum deprivation, they are destined for neuronal death. In this study, we attempted to elucidate the roles of endoplasmic reticular (ER) Ca2+-store and co-cultured astrocytes in HK/serum deprivation induced neuronal death. Thapsigargin (TG), an inhibitor of ER Ca2+-ATPase was simultaneously applied with normal K+ (5 mM K+, NK) serum free medium, and its effects on neuronal death in either astrocyte-poor or astrocyterich culture were examined. By means of the fura-2 microfluorimetric technique, we monitored the changes of the intracellular Ca2+ concentration, [Ca2+]i, associated with neuronal death under various treatments. The results obtained showed that in astrocyte-poor cultures of mature CGN (10 days in vitro, DIV), the basal level of [Ca2+]i markedly decreased from 184 +/- 5 to 89.7 +/- 5 nM 24 h after HK/serum deprivation. Although treatment with TG slightly increased the [Ca2+]i to 117.6 +/- 4 nM, the survival rate of the neurons was even worse; it was reduced from 49 +/- 4% to 28 +/- 2%. In the astrocyte-rich cultures, HK/serum deprivation also caused a profound reduction of neuronal [Ca2+]i, from 166 +/- 3 to 90.2 +/- 6 nM, accompanied by even more serious neuronal death (95.5 +/- 1%). On the other hand, treatment with TG in astrocyterich cultures further lowered the [Ca2+]i to 65 +/- 2 nM but markedly improved the neuronal survival rate from 4.5 +/- 1% to 60 +/- 2% in a concentration-dependent manner. The strong implication of these findings is that ER Ca2+-store and astrocytes participate in modulating the responses of neurons to stress stimulation.

Animals↗

PubMed1999

Nitric oxide prevents apoptosis of human endothelial cells from high glucose exposure during early stage.

Hyperglycemia is a major cause of diabetic vascular disease. High glucose can induce reactive oxygen species (ROS) and nitric oxide (NO) generation, which can subsequently induce endothelial dysfunction. High glucose is also capable of triggering endothelial cell apoptosis. Little is known about the molecular mechanisms and the role of ROS and NO in high glucose-induced endothelial cell apoptosis. This study was designed to determine the involvement of ROS and NO in high glucose-induced endothelial cell apoptosis. Expression of endothelial nitric oxide synthase (eNOS) protein and apoptosis were studied in cultured human umbilical vein endothelial cells (HUVECs) exposed to control-level (5.5 mM) and high-level (33 mM) glucose at various periods (e.g., 2, 12, 24, 48 h). We also examined the effect of high glucose on H(2)O(2) production using flow cytometry. The results showed that eNOS protein expression was up-regulated by high glucose exposure for 2-6 h and gradually reduced after longer exposure in HUVECs. H(2)O(2) production and apoptosis, which can be reversed by vitamin C and NO donor (sodium nitroprusside), but enhanced by NOS inhibitor (N(G)-nitro-L-arginine methyl ether), were collated to a different time course (24-48 h) to HUVECs. These results provide the molecular basis for understanding that NO plays a protective role from apoptosis of HUVECs during the early stage (<24 h) of high glucose exposure, but in the late stage (>24 h), high glucose exposure leads to the imbalance of NO and ROS, resulting to the observed apoptosis. This may explain, at least in part, the impaired endothelial function and vascular complication of diabetic mellitus that would occur at late stages.

Apoptosis↗

PubMed1999

Suramin inhibits the toxic effects of presynaptic neurotoxins at the mouse motor nerve terminals.

Clinically available chemical antagonists of snake neurotoxins still await to be identified. In this study, we demonstrate that an anti-trypanosomiasis agent, suramin, is an effective inhibitor of beta-bungarotoxin isolated from the venom of Formosan Krait snake. Following intraperitoneal injection (12 ng/g) of beta-bungarotoxin in mice, the time to paralysis (loss a limb withdrawal reflex, 21. 8+/-3.4 h, n=4) was significantly prolonged after intravenous injection (16 microg/g) of suramin (35.9+/-4.0 h, n=4, P<0.05). The mechanism of this inhibitory effect of suramin was analyzed at the mouse nerve terminals. beta-Bungarotoxin (1 microg/ml) produces an irreversible blocking effect of nerve-evoked muscle contractions of mouse phrenic nerve-diaphragm (blocking time 135+/-6 min, n=6). Pretreatment with suramin (0.3 mM) significantly prolonged the blocking time by three-fold. This selective inhibitory effect of suramin was further confirmed when suramin was shown to delay the neuromuscular blocking effect of another presynaptic neurotoxin, crotoxin (from American rattlesnake venom), but not that of the postsynaptic neurotoxin, alpha-bungarotoxin. Furthermore, suramin inhibited beta-bungarotoxin in blocking transmitter release as revealed by prolonging the time to abolish the end-plate potential amplitude (with suramin, 391+/-8 min; without treatment, 141+/-5 min). K(+) current was measured in the mouse triangularis sterni preparation; suramin (0.3 mM) had no significant effect on beta-bungarotoxin in inhibiting K(+) current (77+/-3% of control; with suramin 75+/-3% of control, respectively). These findings clearly show that suramin is an inhibitor of presynaptic neurotoxins, mediated by interrupting the toxins in blocking the releasing mechanism of transmitter at the motor nerve terminals. The implication of these findings is that suramin and related compounds can become useful agents in management of snakebites.

Animals↗

PubMed1999

Inhibition of cyclin-dependent kinases 2 and 4 activities as well as induction of Cdk inhibitors p21 and p27 during growth arrest of human breast carcinoma cells by (-)-epigallocatechin-3-gallate.

(-)-Epigallocatechin-3-gallate (EGCG) potently inhibits cell proliferation and suppresses tumor growth both in vitro and vivo, but little is known regarding the cell cycle regulatory proteins mediating these effects. This study investigated the effects of EGCG and other catechins on the cell cycle progression. DNA flow cytometric analysis indicated that 30 microM of EGCG blocked cell cycle progression at G1 phase in asynchronous MCF-7 cells. In addition, cells exposed to 30 microM of EGCG remained in the G1 phase after release from aphidicolin block. Over a 24-h exposure to EGCG, the Rb protein changed from hyper- to hypophosphorylated form and G1 arrest developed. The protein expression of cyclin D1, and E reduced slightly under the same conditions. Immunocomplex kinase experiments showed that EGCG inhibited the activities of cyclin-dependent kinase 2 (Cdk2) and 4 (Cdk4) in a dose-dependent manner in the cell-free system. As the cells were exposed to EGCG (30 microM) over 24 h a gradual loss of both Cdk2 and Cdk4 kinase activities occurred. EGCG also induced the expression of the Cdk inhibitor p21 protein and this effect correlated with the increase in p53 levels. The level of p21 mRNA also increased under the same conditions. In addition, EGCG also increased the expression of the Cdk inhibitor p27 protein within 6 h after EGCG treatment. These results suggest that EGCG either exerts its growth-inhibitory effects through modulation of the activities of several key G1 regulatory proteins such as Cdk2 and Cdk4 or mediates the induction of Cdk inhibitor p21 and p27.

CDC2-CDC28 Kinases↗

PubMed1999

Induction of apoptosis by penta-O-galloyl-beta-D-glucose through activation of caspase-3 in human leukemia HL-60 cells.

Penta-O-galloyl-beta-D-glucose is structurally related to (-)-epigallocatechin gallate and is isolated from hydrolyzed tannin. Penta-O-galloyl-beta-D-glucose can inhibit tumor promotion by teleocidin. We investigated the effects of penta-O-galloyl-beta-D-glucose and various tea polyphenols on cell viability in human leukemia HL-60 cells. In this study, we demonstrated that penta-O-galloyl-beta-D-glucose was able to induce apoptosis in a concentration- and time-dependent manner; however, other polyphenols were less effective. We further investigated the molecular mechanisms of penta-O-galloyl-beta-D-glucose-induced apoptosis. Treatment with penta-O-galloyl-beta-D-glucose caused induction of caspase-3/CPP32 activity in dose- and time-dependent manner, but not caspase-1 activity, and induced the degradation of poly-(ADP-ribose) polymerase. Pretreatment with acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO) and Z-Val-Ala-Asp-fluoromethyl-ketone (Z-VAD-FMK) inhibited penta-O-galloyl-beta-D-glucose-induced DNA fragmentation. Furthermore, treatment with penta-O-galloyl-beta-D-glucose (50 microM) caused a rapid loss of mitochondrial transmembrane potential, release of mitochondrial cytochrome c into cytosol, and subsequent induction of procaspase-9 processing. Our results indicate that penta-O-galloyl-beta-D-glucose allows caspase-activated deoxyribonuclease to enter the nucleus and degrade chromosomal DNA, and induces DFF-45 (DNA fragmentation factor) degradation. These results lead to a working hypothesis that penta-O-galloyl-beta-D-glucose-induced apoptosis is triggered by the release of cytochrome c into the cytosol, procaspase-9 processing, activation of caspase-3, degradation of poly-(ADP-ribose) polymerase, and DNA fragmentation caused by the caspase-activated deoxyribonuclease through the digestion of DFF-45. The induction of apoptosis by penta-O-galloyl-beta-D-glucose may provide a pivotal mechanism for its cancer chemopreventive action.

Acridine Orange↗

PubMed1999

Cancer chemoprevention by tea polyphenols through mitotic signal transduction blockade.

Tea is a popular beverage. The consumption of green tea is associated with a lower risk of several types of cancer, including stomach, esophagus, and lung. The cancer chemopreventive effect of tea has been attributed to its major phytopolyphenols. The tea polyphenols comprise about one-third of the weight of the dried leaf, and they show profound biochemical and pharmacological activities including antioxidant activities, modulation of carcinogen metabolism, inhibition of cell proliferation, induction of cell apoptosis, and cell cycle arrest. They intervene in the biochemical and molecular processes of multistep carcinogenesis, comprising tumor initiation, promotion, and progression. Several studies demonstrate that most tea polyphenols exert their scavenging effects against reactive oxygen species (ROS); excessive production of ROS has been implicated for the development of cardiovascular diseases, neurodegenerative disorders, and cancer. Recently, we have found that the major tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) suppresses extracellular signals and cell proliferation through epidermal growth factor receptor binding in human A431 epidermoid carcinoma cells; EGCG also blocks the induction of nitric oxide synthase by down-regulating lipopolysaccharide-induced activity of the transcription factor NFKB in macrophages. Furthermore, EGCG blocks the cell cycle at the G1 phase in MCF-7 cells. We have demonstrated that EGCG inhibits the activities of cyclin-dependent kinases 2 and 4; meanwhile, EGCG induces the expression of the Cdk inhibitors p21 and p27. These results suggest that tumor promotion can be enhanced by ROS and oxidative mitotic signal transduction, and this enhancement can be suppressed by EGCG or other tea polyphenols.

Cell Transformation, Neoplastic↗

PubMed1999

Suppression of TNFalpha-mediated NFkappaB activity by myricetin and other flavonoids through downregulating the activity of IKK in ECV304 cells.

Flavonoids are a group of naturally-occurring phenolic compounds in the plant kingdom, and many flavonoids are found with vascular protective properties. Nevertheless how the protective response is exerted by flavonoids is not well characterized. In view of the nuclear factor-kappaB (NFkappaB) may play a central role in the initiation of atherosclerosis, prevention of the activation of NFkappaB represents an important role in protecting vascular injury. In this study, the effects of flavonoids on NFkappaB/inhibitor-kappaB (IkappaB) system in ECV304 cells activated with tumor necrosis factor-alpha (TNFalpha) were examined. We investigated the inhibitory action of six flavonoids on IkappaB kinase (IKK) activity, an enzyme recently found to phosphorylate critical serine residues of IkappaB for degradation. Of six flavonoids tested, myricetin was found to strongly inhibit IKK kinase activity, and prevent the degradation of IkappaBalpha and IkappaBbeta in activated endothelial cells. Furthermore, myricetin was also found to inhibit NFkappaB activity correlated with suppression of monocyte adhesion to ECV304 cells. Therefore we conclude that flavonoids may be of therapeutic value for vascular disease through down regulation of NFkappaB/IkappaB system.

Base Sequence↗

PubMed1999

Role of tyrosine kinase activity in 2,2',2''-tripyridine-induced nitricoxide generation in macrophages.

In this paper, we demonstrated that 2,2',2"-tripyridine (TP, 1-20 microM) is a potent inducer of nitric oxide (NO) synthase in the cultured murine macrophage RAW 264.7 cell line. TP increased not only nitrite but also inducible NO synthase (iNOS) protein and mRNA production. Co-treatment with either NOS inhibitors (N(G)-monomethyl-L-arginine and aminoguanidine) or cycloheximide and actinomycin D all inhibited TP-induced nitrite production, indicating the requirement of protein and mRNA synthesis. The signaling pathway of TP-induced iNOS expression was explored, and the results obtained suggested that increased tyrosine kinase activity followed by inhibitor of nuclear factor for immunoglobulin kappa chain in B cells (IkappaB) degradation and then nuclear factor kappaB (NFkappaB) activation was involved in TP-induced iNOS expression. Tyrosine kinase inhibitors (e.g. genistein and tyrphostin AG126) inhibited both TP-induced nitrite and iNOS protein production. Whether the metalochelating property of TP was involved in these effects was explored by saturating TP with FeCl3. Although the ferrated TP became inactive, the specific iron chelator desferrioxamine, at a very high concentration of 400 microM, induced only a weak enhancement of nitrite production in this RAW cell line. It was thereby concluded that TP induces NO production through an increase in iNOS expression, which is initiated by a signaling pathway via tyrosine kinases leading to an activation of NFkappaB. Since TP is much more potent than desferrioxamine in increasing nitrite production, it is suspected that the primary event induced by TP was possibly mediated by TP's interacting with certain macromolecules in addition to its metal-chelating property.

Animals↗