The component of green tea,L-theanine protects human hepatic L02 cells from hydrogen peroxide-induced apoptosis

L-theanine is a natural amino acid in green tea and it has been well known for its activities of relieving depression and neuroprotection. However, cytoprotective effect and its mechanism of L-theanine on hepatocytes have not been reported. The objective of this work was to investigate the hepatoprotective effect of L-theanine as well as its mechanism by using the human hepatic L02 cells injured by hydrogen peroxide (H₂O₂). Results showed that L-theanine dose dependently decreased H₂O₂-induced cell viability loss and lactate dehydrogenase (LDH) release. L-theanine pretreatment improved nuclear morphology of the cells injured by H₂O₂. By using flow cytometric analysis, we found that L-theanine significantly inhibited H₂O₂-induced cell apoptosis. Further, L-theanine attenuated H₂O₂-induced reduction in pro-caspase3 and cleavage of poly (adenosine diphosphate-ribose) polymerase (PARP). H₂O₂-activated p38 mitogen-activated protein kinase (MAPK) was also inhibited by L-theanine. These data suggest that L-theanine could protect L02 cells against H₂O₂-induced apoptosis via suppression of p38 MAPK. L-theanine might potentially be useful in the prevention and treatment of liver diseases.     

Acanthoic acid induces cell apoptosis through activation of the p38 MAPK pathway in HL-60 human promyelocytic leukaemia

The present study was designed to evaluate the molecular mechanisms of the action of acanthoic acid (ACAN) from Acanthopanax koreanum (Araliaceae) against HL-60 human promyelocytic leukaemia cells. ACAN reduced the proliferation of HL-60 cells in a dose- and time-dependent manner accompanied by the induction of apoptosis. Possible mechanisms of ACAN-induced apoptosis were also examined. The results showed that ACAN-induced the phosphorylation of members of the mitogen-activated protein kinase (MAPK) family, c-Jun N-terminal kinase (JNK), p38 MAPK (p38), and extracellular signal-regulated kinase (ERK). A specific p38 MAPK inhibitor (SB203580) significantly blocked ACAN-induced apoptosis and cell viability, whereas an ERK inhibitor (PD98059) and JNK inhibitor (SP600125) had no effect. Moreover, ACAN induced the cleavage of caspase-3 and poly-ADP-ribose polymerase (PARP), and decreased the level of Bcl-xL, but these effects were inhibited by SB203580 pre-treatment. These results strongly suggest that ACAN may have cancer chemopreventive and therapeutic potential, due to its ability to activate the p38 MAPK-mediated signalling pathways.     

Hempseed protein derived antioxidative peptides: Purification,identification and protection from hydrogen peroxide-induced apoptosis in PC12 cells

Hempseeds have not been utilised to any significant extent by the industrial food markets. A novel antioxidant peptide derived from hempseed by-product was investigated. Hempseed protein isolate was prepared by alkaline extraction and subsequent isoelectric precipitation. Its enzymatic hydrolysate, obtained by Alcalase® treatment, was subjected to DA201-C macroporous absorption resin, with simultaneous desalting and concentrating of hydrophobic fragments with improved free radical-scavenging activities. The active fraction was further separated by gel filtration and reversed-phase high performance liquid chromatography to obtain two purified peptides; the peptides were characterised as NHAV and HVRETALV, by MALDI-TOF/TOF MS/MS. The protective effects of the purified peptides on hydrogen peroxide-induced cell apoptosis were investigated in rat pheochromocytoma line PC12 cells. The peptides, at a concentration of 10 μg/ml, possessed protective effects (P < 0.05) against cell death and oxidative apoptosis. The findings are significant for the discovery and development of natural antioxidants from hempseed by-products.     

Changes in apoptosis factors and activation of caspase-3 in tilapia muscle during storage

The activation of the apoptosis pathway in tilapia muscle during postmortem storage was studied. Changes in caspase-3 activity, ATP content, cytochrome c levels, and ratio of Bcl-2/Bax levels of tilapia muscle were observed during postmortem storage at 20°C. Caspase-3 activity was found to be significantly increased at first, followed by a decrease (P < 0.05); the highest caspase-3 activity was observed at 1 h. The ATP content decreased significantly (P < 0.05), and almost exhausted after 10 h storage. The cytochrome c level in the cytosol showed a significant increase after 5 h of storage (P < 0.05), while the mitochondrial cytochrome c levels showed a decrease. The Bcl-2/Bax ratio was stable from 0–5 h, followed by a rapid decreased at 10–20 h and a significant increased after 20 h (P < 0.05), suggesting that the apoptosis process occurred until 20 h of postmortem storage. Thus, we concluded that the availability of ATP and the increase in cytosolic cytochrome c levels are essential for the activation of caspase-3, and that the former partly limits caspase-3 activity.     

p38 MAPK activation, DNA damage, cell cycle arrest and apoptosis as mechanisms of toxicity of silver nanoparticles in Jurkat T cells

To identify potential harmful effects of silver nanoparticles (AgNPs) on human health, a comprehensive toxicity assay was conducted on human Jurkat T cells, using oxidative stress-related endpoint. The effect of Ag ions was also investigated and compared with that of AgNPs, as it is anticipated that Ag ions will be released from AgNPs, which may be responsible for their toxicity. Cell viability tests indicated high sensitivity of Jurkat T cells when exposed to AgNPs compared to Ag ions; however, both AgNPs and Ag ions induce similar levels of cellular reactive oxygen species during the initial exposure period and; after 24 h, they were increased on exposure to AgNPs compared to Ag ions, which suggest that oxidative stress may be an indirect cause of the observed cytotoxicity of AgNPs. AgNPs exposure activates p38 mitogen-activated protein kinase through nuclear factor-E2-related factor-2 and nuclear factor-kappaB signaling pathways, subsequently inducing DNA damage, cell cycle arrest and apoptosis. Selective toxicity of AgNPs on Jurkat T cells suggests that rigorous toxicity evaluation should be conducted using various different cell types and biological systems prior to the widespread use of AgNPs.     

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.     

Carbonyl compounds methylglyoxal and glyoxal affect interleukin‐8 secretion in intestinal cells by superoxide anion generation and activation of MAPK p38

The carbonyl compounds methylglyoxal (MG) and glyoxal (GL) are reactive intermediates of glucose degradation pathways and capable of inducing cellular damage. Although immune‐stimulating activity has been investigated in endothelial cells, little is known about the signaling pathways of cytokine induction of these compounds in the intestine. Hence, we investigated the impact of mitogen‐activated protein kinases (MAPK) and nuclear factor kappa B (NF‐κB) on IL‐8 production by human intestinal cells (Caco‐2 and HT‐29) after stimulation by MG and GL. Both compounds induced a dose‐dependent enhancement of IL‐8 secretion in human intestinal cells. MAPK p38 and extracellular signal‐regulated kinase (ERK) were phosphorylated in these cells after having been stimulated by MG and GL. Furthermore, inhibitors of MAPK p38 (SB 203580 and 239063), ERK1/2 (PD 98059) and NF‐κB activation (SM‐7368 and SC‐514) reduced IL‐8 secretion. The most important mechanism by which MG and GL induced IL‐8 secretion was the generation of superoxide anions which was confirmed by the inhibition of the cytosolic NADPH oxidase with diphenyl iodonium (DPI) or by application of superoxide dismutase (SOD). Our data suggest that multiple pathways were simultaneously activated; however, superoxide dependent MAPK p38 activation seems to be the most dominant pathway for IL‐8 secretion in intestinal cells.     

Xanthohumol induces apoptosis in cultured 40-16 human colon cancer cells by activation of the death receptor- and mitochondrial pathway

Xanthohumol (XN) is one of the major prenylflavonoids found in hop cones (Humulus lupulus L.). In this study, we investigated the cell growth inhibitory potential of XN on cultured human colon cancer cells. Cell proliferation was measured by sulforhodamine B staining. Poly(ADP-ribose)polymerase (PARP) cleavage, activation of caspases-3, -7, -8, and -9, and Bcl-2 family protein expression were detected by Western blot analyses. XN significantly reduced proliferation of the HCT 116-derived colon cancer cell line 40--16. Half-maximal inhibitory concentrations decreased from 4.1 microM after 24 h treatment to 3.6 and 2.6 microM after 48 and 72 h incubation, respectively. Treatment with 15 microM XN for 48 h and with 5 microM for 72 h led to the detection of the cleaved 89 kDa fragment of 116 kDa PARP as an indication of apoptosis induction. Concomitantly, we observed activation and cleavage of the effector caspases-3 and -7, induced by activation of the initiator caspases -8 and -9. Expression of anti-apoptotic Bcl-2 was down regulated when the cells were treated with XN for 48--72 h. We conclude that induction of apoptosis by downregulation of Bcl-2 and activation of the caspase cascade may contribute to the chemopreventive or therapeutic potential of XN.     

Cross talk between cAMP and p38 MAPK pathways in the induction of leptin by hCG in human placental syncytiotrophoblasts

Leptin produced by the placental syncytiotrophoblasts participates in a number of processes in pregnancy including implantation, proliferation of the cytotrophoblasts, and nutrient transfer across the placenta. Despite the functional significance of leptin in pregnancy, the regulation of leptin synthesis is poorly understood in human placental syncytiotrophoblasts. In this study, we investigated the role of endogenous human chorionic gonadotropin (hCG) in the regulation of leptin production as well as the underlying mechanism involving the cross talk between cAMP and p38 mitogen-activated protein kinase (MAPK) pathways. We found that neutralization of endogenous hCG with its antibody dose dependently decreased leptin mRNA level and secretion, whereas exogenous hCG increased leptin mRNA level and secretion. Activation of the cAMP pathway with dibutyryl cAMP (db cAMP) or forskolin recapitulated the stimulatory effect of hCG on leptin expression. Inhibition of protein kinase A with H89 not only reduced the basal leptin expression but also attenuated the induced leptin expression by hCG. Treatment of the syncytiotrophoblasts with db cAMP and hCG phosphorylated p38 MAPK. Inhibition of p38 MAPK with SB203580 not only reduced the basal leptin production but also attenuated the leptin-induced production by both hCG and db cAMP. These data suggest that endogenous hCG plays a significant role in maintaining leptin production in human placental syncytiotrophoblasts, and this effect involves a cross talk between cAMP and p38 MAPK pathways.     

X-linked inhibitor of apoptosis protein and active caspase-3 expression patterns in antral follicles in the sheep ovary

X-linked inhibitor of apoptosis protein (XIAP) interacts with caspases to inhibit their activity, thereby providing a potential mechanism for regulation of granulosa cell apoptosis occurring during follicular atresia. The aim of this study was to determine the presence and localization of XIAP mRNA and protein content in the sheep ovary and compare these expression patterns with active caspase-3 protein in the same antral follicles. Romney ewe estrous cycles (n=25) were synchronized with 2-3 Estrumate injections and ovarian tissue collected during the luteal and follicular phases of the cycle. The presence of XIAP mRNA was confirmed by RT-PCR using laser capture microdissected ovarian cell samples. XIAP mRNA was subsequently localized by in situ hybridization histochemistry and XIAP and active caspase-3 protein visualized by immunohistochemistry. In antral follicles extensive XIAP localization was evident in both granulosa and thecal cells. In contrast, mRNA expression was widespread in granulosa cells and only detected in thecal tissue from a small proportion of antral follicles. Active caspase-3 and XIAP comparative expression analysis showed positive XIAP mRNA expression in all late luteal phase (day 14) follicles, despite varying levels of active caspase-3 protein. A proportion of follicular phase (days 15 and 16) follicles, however, showed an inverse expression relationship at the protein and mRNA levels in both granulosa and thecal tissue, as did XIAP protein in day 14 follicles. These results suggest high XIAP may prevent activation of caspase-3, thereby regulating follicular atresia in antral follicles and could potentially be utilized as a marker of follicular health.     

EGCG inhibits protein synthesis,lipogenesis, and cell cycle progression through activation of AMPK in p53 positive and negative human hepatoma cells

In the previous studies, (–)‐epigallocatechin‐3‐gallate (EGCG) has been shown to have anticarcinogenic effects via modulation in protein expression of p53. Using p53 positive Hep G2 and p53 negative Hep 3B cells, we found that treatment of EGCG resulted in dose‐dependent inhibition of cellular proliferation, which suggests that the interaction of EGCG with p53 may not fully explain its inhibitory effect on proliferation. Caloric restriction (CR) reduces the incidence and progression of spontaneous and induced tumors in laboratory rodents. EGCG has multiple beneficial activities similar to those associated with CR. One key enzyme thought to be activated during CR is AMP‐activated kinase (AMPK), a sensor of cellular energy levels. Here, we showed that EGCG activated AMPK in both p53 positive and negative human hepatoma cells. The activation of AMPK suppressed downstream substrates, such as mammalian target of rapamycin (mTOR) and eukaryotic initiation factor 4E‐binding protein‐1 (4E‐BP1) and a general decrease in mRNA translation. Moreover, EGCG activated AMPK decreases the activity and/or expression of lipogenic enzymes, such as fatty acid synthase (FASN) and acetyl‐CoA carboxylase (ACC). Interestingly, the decision between apoptosis and growth arrest following AMPK activation is greatly influenced by p53 status. In p53 positive Hep G2 cells, EGCG blocked the progression of cell cycle at G1 phase by inducing p53 expression and further up‐regulating p21 expression. However, EGCG inducted apoptosis in p53 negative Hep 3B cells. Based on these results, we have demonstrated that EGCG has a potential to be a chemoprevention and anti‐lipogenesis agent for human hepatoma cells.     

Anticancer effects of maple syrup phenolics and extracts on proliferation,apoptosis, and cell cycle arrest of human colon cells

The antiproliferative effects of Canadian maple syrup (grades C and D) extracts and fifty-one purified phenolic constituents were evaluated against human tumourigenic (HT-29, HCT-116, and CaCo-2) and non-tumourigenic (CCD-18Co) colon cells. Overall, maple syrup ethyl acetate (MS-EtOAc), butanol (MS-BuOH), and methanol (MS-MeOH) extracts were more active against the tumourigenic versus non-tumourigenic colon cells. At equivalent phenolic levels, the antiproliferative activities of grade D > C maple syrup, and MS-BuOH > MS-MeOH > MS-EtOAc. Among the isolates, gallic acid, catechaldehyde, syringaldehyde, and catechol were most active and their higher levels in grade D MS-BuOH extract could account for the highest observed anticancer effects of that extract. Moreover, the maple syrup extracts did not induce apoptosis of the colon cancer cells but induced cell cycle arrest which was also associated with a decrease in cyclins A and D1 levels. These results suggest that phenolics may impart potential biological effects to maple syrup.