Immunomodulatory effects of the pentapeptide YGSRS on human peripheral-blood mononuclear cells.

CONTEXT: The pentapeptide YGSRS is originated from coffee bean, while its pharmacological features have little been examined. OBJECTIVES: We investigated the effects of YGSRS on proliferation, cytokine production and CD4+ CD25+ Foxp3+ regulatory T (Treg) cell frequency of human peripheral blood mononuclear cells (PBMCs) activated by T-cell mitogen. MATERIALS AND METHODS: The effects of YGSRS on T-cell mitogen-activated PBMCs were assessed by WST assay procedures. Concentrations of Th1/Th2/Th17 cytokines in the PBMCs culture medium were analyzed with beads-array procedures followed by analysis with flow cytometry. The CD4+ CD25+ Foxp3+ Treg cells in mitogen-activated PBMCs were stained with fluorescence-labeled specific antibodies followed by flow cytometry. RESULTS: YGSRS at 1-10,000 ng/ml (1.56-15,600 nM) has a tendency to promote the mitogen-activated proliferation of PBMCs, but the effects were not statistically significant. YGSRS affect the production of tumor necrosis factor (TNF) α, interleukin (IL)-4, IL-6 and IL-10 from the activated PBMCs, and statistically significant increase in the concentrations of IL-6 and IL-10 in the medium were observed at 1-1000 ng/ml (1.56-1560 nM) (p < 0.05). YGSRS has a tendency to decrease the frequency of Treg cells in the activated PBMCs, but the difference was not statistically significant. DISCUSSION AND CONCLUSIONS: The data suggest that the pentapeptide YGSRS affects the production of several types of cytokines from activated human peripheral T cells, which may modulate Th2 type immunity.

Hydroxyapatite-coated titanium oxide ameliorates dextran sulphate sodium-induced colitis by attenuating both innate and acquired immune reaction.

Introduction: Titanium oxide (TiO2) is a widely used oxidizer for environmental management. The power of TiO2 has been demonstrated by its photocatalytic activity. Hydroxyapatite (HA)-coated TiO2 (HA-TiO2) was used to test the in vivo effect on dextran sulphate sodium (DSS)-induced colitis in mice. Material and methods: Mice were monitored for body weight and then sacrificed on the seventh day, and the colon length was measured. Their faeces were analysed for intestinal microbiota distribution, and colon tissue was subjected to histological examination and immunohistochemical analysis. Results: Weight loss was significantly lower in HA-TiO2-fed mice than in mice without HA-TiO2. The colon length in the DSS colitis-induced mice was shortened, but HA-TiO2 feeding lessened this effect. Histological and immunohistochemical analyses of the colon revealed that macrophages and CD4+CD8+ T cells were observed in the colitis-occurring site, indicating the involvement of innate and acquired immunity in determining the degree of DSS-induced colitis. Intestinal microbiota analysis in faeces revealed changes in the distribution of multiple bacterial species after DSS colitis induction, and the increase/decrease of 2 Clostridium (sub)clusters moved in response to the colitis phenomenon. All the described effects of HA-TiO2 were photocatalytic activity-dependent because mice that were kept in the dark showed similar results to those treated with DSS alone without HA-TiO2. Conclusions: HA-coated TiO2 ameliorated DSS-induced colitis through photocatalytic activity, while HA-TiO2 diminished the changes in intestinal microbiota and immune reactions caused by DSS.

Cladribine enhances apoptotic cell death in lung carcinoma cells over-expressing DNase γ.

Worldwide, lung cancer is the most common form of cancer and often has a poor prognosis. Establishment of effective therapies for lung cancer is a major concern in clinical cancer research. We compared the cytotoxic effects of chemotherapeutic agents including cisplatin, 5-fluorouracil, vinorelbine and cladribine, on a human lung cancer cell line, A549, and its derivative transfected with the DNase γ gene. We observed selective cytotoxicity of cladribine on the DNase γ-expressing sub-cell line of A549. Cladribine induces selective apoptosis in DNase γ-expressing A549 cells, which depends on activation of caspases. These results suggest that a combination therapy that includes cladribine along with the introduction of DNase γ has potential as a new therapeutic strategy for lung cancer.

Free calvarial periosteum graft vascularized by an omental flap in a rat model.

Because omental flaps are useful for flap prefabrication and the cambium layer of the periosteum can be osteogenic, we examined whether calvarial periosteum grafted onto greater omentum of rats was osteogenic and suitable for a flap. Distal omentum was wrapped with calvarial periosteum and so the cambium faced the omentum. Grafted omentum was harvested at 1 to 9 days. In other rats, grafted omentum was elevated as a pedicled flap and moved to the abdominal subcutis, to be harvested later at 1 to 5 months after the initial surgery. Bone formation was evaluated histologically, histochemically, and radiographically. On day 3, osteoid had formed. From day 4, calvarial periosteum was revascularized by omentum and bone was forming. New bone was maintained after grafting to subcutis for 5 months. Thus, bone formed by periosteum on the omentum could be used to reconstruct defects of the bone.

Omental flap closure of refractory wounds: rat model.

Omental flaps, with their associated rich and pliable vascular arcades, are frequently used in clinical practice for the reconstruction of complex and irregular defects. There is little experimental evidence, however, to prove that omental flaps can be a useful tool for the defects. Using a gastric-wall defect model, we performed histological and immunocytochemical examinations. We created an omental flap lining a 2.0-mm defect perforating the center of the anterior wall of a rat stomach. We examined the tissue response during gastric wall regeneration by H&E and Masson trichrome stains. We also performed immunocytochemical studies for the detection of proliferating cell nuclear antigen (PCNA), factor VIII-related antigen, fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factor (VEGF). One day after the operation, the omental flap was found to firmly adhere to the gastric serosa surrounding the defect. An extensive inflammatory response occurred from Day 1 to 3 with dilated vessels in the omentum. From Days 3 to 7, a significant number of PCNA-positive cells, FGF-2-positive cells and VEGF-positive cells were observed at the edge of the mucosa and within the granulation tissue. On Day 4, in place of extensive inflammation, an exuberant granulation tissue response was observed from the omentum. The defect had been covered by stratified villi by Day 7. This study demonstrated that an omental flap came to rapidly adhere to the defect serving as a source of extensive inflammation and granulation for the rich and pliable vascular arcades.

Phosphatidylcholine induces growth inhibition of hepatic cancer by apoptosis via death ligands.

BACKGROUND/AIMS: Phosphatidylcholine reduces chemically-induced hepatocarcinogenesis in rats and the growth of hepatic cancer cells. We planned to determine whether apoptosis pathways via death ligands were induced by phosphatidylcholine. METHODOLOGY: Growth inhibition of hepatic cancer cell lines (HepG2, Hep3B, Alexander, and HuH-7) was examined by MTT assay. On apoptosis induction, flow cytometry analyses were performed after Fas or TNF-alpha ligand stimulation followed by phosphatidylcholine. Expressions of caspase-8, -3 and PARP after phosphatidylcholine stimulation were examined by immunoblotting. TUNEL staining was also performed after phosphatidylcholine stimulation. RESULTS: MTT assays showed growth inhibitions by phosphatidylcholine dose-dependently. Ratios of sub-G1 phase cell population by FACScan significantly increased on 48h phosphatidylcholine stimulation in comparison to the control group (p < 0.05). Fas or TNF-alpha ligand followed by phosphatidylcholine stimulation significantly increased apoptotic cells more than by phosphatidylcholine alone (p < 0.05). Enhanced appearances of cleaved caspase-8, -3 and fragmented PARP were shown on immunoblotting and apoptotic cells on TUNEL staining after phosphatidylcholine stimulation. Phosphatidylcholine was assumed to reduce hepatic carcinogenesis by apoptosis induction via the death ligands (Fas and/or TNF-alpha) pathway followed by caspase-8 and -3 inductions. CONCLUSIONS: Phosphatidylcholine intake may well inhibit carcinogenesis in patients at high risk for hepatocellular carcinoma by apoptosis induction.

Bid truncation mediated by caspases-3 and -9 in vinorelbine-induced apoptosis.

Vinorelbine is a chemotherapeutic vinca alkaloid clinically prescribed for non-small cell lung cancer and breast cancer. Here we studied the mechanism for vinorelbine-induced apoptosis in a human T-cell lymphoma. Although vinorelbine induces DNA fragmentation that is inhibited by specific peptide inhibitors for caspases-9 and -3 in Jurkat cells, caspase-8 deficiency retards vinorelbine-induced apoptosis. Activation of caspase-8 is also observed in vinorelbine-treated cells, and the activity is diminished when the caspase-3 activity is blocked by a specific peptide inhibitor, Ac-DNLC-CHO. Blocking of the Fas receptor with an antagonistic anti-Fas antibody does not affect vinorelbine-induced DNA fragmentation. These results suggest that vinorelbine-induced apoptosis is enhanced by the activation of caspase-8 via caspase-9-mediated activation of caspase-3, but not through a Fas-triggered signal. Western blotting suggests that vinorelbine cleaves caspase-3, -9 and -8 and reduces the amount of mitochondrial cytochrome c. Caspase-8 deficiency suppresses all of these events. A downstream substrate for caspase-8, Bid, is also cleaved in vinorelbine-treated cells, but the Bid truncation is also observed in caspase-8-deficient Jurkat cells. Importantly, recombinant caspases-3 and -9, as well as caspase-8, directly cleaves recombinant Bid in vitro. These results suggest that caspases-3 and -9 participate in Bid truncation, indicating a new mechanism for vinorelbine-induces apoptosis.

Chemo-sensitivity of Two-dimensional Monolayer and Three-dimensional Spheroid of Breast Cancer MCF-7 Cells to Daunorubicin, Docetaxel, and Arsenic Disulfide.

BACKGROUND/AIM: Chemo-sensitivity of two-dimensional (2D) monolayers and three-dimensional (3D) spheroids of human breast cancer MCF-7 cells were investigated. MATERIALS AND METHODS: MCF-7 cells were cultured in monolayers or spheroids established using a thermo-reversible gelatin polymer, in the presence of daunorubicin, docetaxel, or As2S2 Cell proliferation was examined by a Cell Counting Kit-8 assay. RESULTS: Daunorubicin, docetaxel, and As2S2 dose-dependently decreased the MCF-7 cell proliferation in both 2D- and 3D-culture systems. The 3D spheroids were less sensitive to these agents than the 2D cultured cells. Verapamil, an inhibitor of P-glycoprotein, partially enhanced the antiproliferative effects of the agents. DL-buthionine-(S, R)-sulfoximine significantly increased (p<0.05), while N-acetyl-L-cysteine significantly inhibited the antiproliferative effects of As2S2 (p<0.003). CONCLUSION: The 3D spheroids showed less sensitivity to the antiprolliferative efficacies of anticancer agents than the 2D cultured cells. P-Glycoprotein is suggested to be partially implicated in drug resistance. Reduction of cellular glutathione level enhanced the As2S2 cytotoxicity.

[Apoptotic mechanisms induced in breast cancer cells by vinorelbine].

Vinorelbine (VNB) is one of new semi-synthesized vinka alkaloids developed in France, of which anti-tumor activity is susceptible mainly to non-small cell lung cancer and breast cancer. Moreover, its clinical efficacy has been noted from single-agent therapy or combination therapy with taxanes. It is assumed that VNB selectively acts on tubulin which elaborates microtubules, strands the cells at G 1 phase and interferes with the mitosis. VNB has unique anti-tumor activity as an antimicrotubule agent and is expected to be available for treatment of multi-drug resistant tumors. In this report, we demonstrate that VNB, as an antimicrotubule agent, induces apoptosis in breast cancer cell line, MX-1 via a mitochondrial pathway.

Gene therapy for hepatocellular carcinoma using sonoporation enhanced by contrast agents.

We examined whether sonoporation enhanced by a contrast agent (BR14) was effective in gene therapy for hepatocelluar carcinoma (HCC). Human hepatic cancer cells (SK-Hep1) and plasmid cDNAs expressing green fluorescent protein (GFP), interferonbeta (IFNbeta), and LacZ were used. In vitro, SK-Hep1 cell suspensions with DNA and BR14 were sonoporated. Expressions of every plasmid cDNA and the antitumor effect of IFNbeta were analyzed. In vivo, GFP and IFNbeta genes with BR14 were directly injected into subcutaneous tumors using SK-Hep1 in nude mice, and transcutaneous sonoporation of the tumors was performed. GFP gene transfections and tumor diameters after IFNbeta gene transfection were examined. In vitro, no SK-Hep1 cells were transfected without sonication, whereas transfections were successful after sonication with BR14. Antitumor effect of IFNbeta gene transfection by ultrasound (US) and with BR14 was revealed. In vivo, the SK-Hep1 cells expressed GFP, and the IFNbeta gene transfection by US with BR14 reduced tumor size significantly. In conclusion, gene therapy with sonoporation enhanced by a contrast agent may become a new treatment option for HCC.

Involvement of MKK6 in TCRalphabeta(int)CD69lo: a target population for apoptotic cell death in thymocytes.

By analyzing real-time caspase activity and DNA fragmentation in live thymocytes, we found that apoptosis occurs predominantly in a TCRalphabeta(int)/hiCD69lo population. The number of caspase-active cells and DNA-fragmented cells in MKK6-deficient mice, which were originally generated in our laboratory by gene targeting, was decreased in the TCRalphabeta(int)CD69lo population but not in the TCRalphabetahiCD69lo population. The percentage of caspase-active cells in the H-Y-specific TCRint population was more clearly decreased in male MKK6-deficient H-Y TCR-transgenic mice. Furthermore, the absolute number of TCRhiCD4loCD8lo cells, which are developmentally next to TCRintCD4hiCD8hi cells, was increased in MKK6-deficient H-Y TCR-transgenic mice. Deletion of TCRalphabeta(int)CD4hiCD8hi cells by injecting antigenic lymphocytic chorio-meningitis virus (LCMV) peptide into LCMV-specific TCR-transgenic mice was incomplete in MKK6-deficient mice. Cellular death of TCRalphabeta(int) fetal thymocytes induced by adding an antigenic peptide into an in vitro fetal thymic organ culture system was also diminished in MKK6-deficient TCR-transgenic thymi. These results indicate that MKK6 plays a role in the developing thymocytes, especially in the population of TCRalphabeta(int)CD69lo cells, which possibly undergo negative selection.

Prevention of hepatocarcinogenesis with phosphatidylcholine and menaquinone-4: in vitro and in vivo experiments.

BACKGROUND/AIMS: We examined whether phosphatidylcholine inhibited growth of hepatic cancer, as previously shown for menaquinone-4 (vitamin K2). METHODS: Growth inhibitions by phosphatidylcholine and/or menaquinone-4 and apoptosis induction by phosphatidylcholine were evaluated in vitro using human hepatic cancer cell lines (Hep-3B, Hep-G2, HuH-7, and Alexander). Effects of these agents were then investigated in male Sprague-Dawley rats against hepatocarcinogenesis induced by diethylnitrosamine plus phenobarbital. All rats were killed to examine livers to evaluate inhibitory potential macroscopically and immunohistochemically using an antibody against the marker of carcinogenesis, glutathione S-transferase and apoptotic induction by phosphatidylcholine using TUNEL staining. Blood samples were obtained by cardiac puncture. RESULTS: In vitro, phosphatidylcholine and menaquinone-4 each inhibited cancer cell growth and phosphatidylcholine induced apoptosis dose-dependently. Moreover, exposure to both synergistically inhibited growth in Hep-3B. In vivo, diets containing phosphatidylcholine with or without menaquinone-4 significantly reduced the number of macroscopic hepatic tumor nodules and the extent of abnormally immunoreactive foci conserving hepatic function on serum examinations compared with controls given only the carcinogens. Moreover, phosphatidylcholine supplementation induced apoptosis on TUNEL staining of liver sections. CONCLUSIONS: Given together, phosphatidylcholine and menaquinone-4 may exhibit synergy against hepatocarcinogenesis conserving hepatic function that could benefit patients at high risk for hepatocellular carcinoma.

Intracellular signaling in the induction of apoptosis in a human breast cancer cell line by water extract of Mekabu.

BACKGROUND: We previously reported that water extract of Mekabu, a kind of seaweed, induced apoptosis in a human breast cancer cell line. In the present study we investigated intracellular signaling in apoptosis, with a focus on caspases. METHODS: Mekabu extract, obtained with ultrapure water, was used to induce apoptosis in a human breast cancer cell line, MDA-MB231, and DNA fractionation was investigated by flow cytometry and electrophoresis. In addition, using the caspase detection kit Caspa Tag, activation of caspases 3, 6, 8, and 9 was observed under a fluorescence microscope. Furthermore, using antibodies to caspases 3, 8, 9, and Bid, we conducted a protein analysis by Western blotting to determine the activation of these substances. RESULTS: Obvious ladder formation demonstrating DNA fractionation was seen, confirming that Mekabu extract induced apoptosis. In the fluorescence microscope observations, activation of caspases 3, 6, and 8, but not caspase 9, was seen. Activated caspases 3 and 8 were detected in the Western blotting analysis, but no proteins of activated caspase 9 or Bid were detected. CONCLUSION: Mekabu extract activates caspases 3, 6, and 8 and contributes to intracellular signaling to induce apoptosis in a human breast cancer cell line. This signaling is not via the mitochondria.

Murine lymph node-derived stromal cells effectively support survival but induce no activation/proliferation of peripheral resting T cells in vitro.

Little is known about the homeostatic mechanisms by which the levels of peripheral lymphocytes are maintained. The survival of naïve T cells in vivo must be maintained by some factors that have not been characterized in an in vitro culture system. In this study, we established a culture system of stromal cells derived from murine lymph nodes and investigated the action of the stromal cells in supporting the survival of resting T cells in vitro. Most of the T cells cocultured with the stromal cells did not die, and the supernatant of cultured stromal cells increase the viability of T cells. This T-cell survival-supporting activity was maintained for more than 7 days. Although interleukin (IL)-4, IL-6, IL-7, and interferon-beta also rescued peripheral T cells from spontaneous cell death, medium-soluble and heat-sensitive factor(s) derived from the stromal cells supported the survival of T cells more effectively and for a longer time than did these cytokines. T cells maintained in the culture system with the stromal cells appeared to remain in a resting G0/G1 state and did not show remarkable DNA synthesis. From these results, it is presumed that some soluble factor(s) other than the tested cytokines that have been identified as supporting T-cell survival are produced from lymph node stromal cells. These factor(s) play an important role in maintenance of resting T cells.

Prevention of free-radical induced apoptosis by induction of human recombinant Cu, Zn-SOD in pig endothelial cells.

Vascular endothelial cells are the prime target in ischemia reperfusion injury. Growing evidence has shown that one of the main etiologies is considered to be reactive oxygen species (ROS) that induce endothelial-cell death either by necrosis or apoptosis. Cultured porcine endothelial cells were transfected with human copper, zinc-superoxide dismutase (h-Cu, Zn-SOD) to investigate whether these cells can prevent apoptosis from oxidative injury in vitro. The endothelial cells were cultured with SIN-1 (3-morpholinosydnonimine-N-ethylcarbanride) as a donor of peroxinitrite (ONOO(-)). The control cells without the gene transfection developed characteristic apoptotic changes both morphologically and biochemically when they were incubated with SIN-1 of 200 M. However, the cells showed necrosis predominantly when the concentration of SIN-1 was 1,000 M. On the other hand, the cells transfected with h-Cu, Zn-SOD showed significantly less evidence of apoptotic change after exposure to SIN-1. Nitric oxide (NO) did not significantly affect the viability of either the control cells or the transfected cells. One of the potent ROS, peroxinitrite, is considered to play a significant role in ischemia reperfusion injury. SIN-1 can produce peroxinitrite in vitro that induces endothelial-cell damage by apoptosis. This type of cytotoxicity can be successfully prevented by transfection of the h-Cu, Zn-SOD into the cells.

Nicorandil inhibits oxidative stress-induced apoptosis in cardiac myocytes through activation of mitochondrial ATP-sensitive potassium channels and a nitrate-like effect.

The anti-anginal drug nicorandil has been shown to inhibit apoptosis by activating mitochondrial ATP-sensitive potassium (K(ATP)) channels. The possible contribution of the nitrate moiety of this drug to its anti-apoptotic effect has now been investigated in neonatal rat ventricular myocytes subjected to oxidative stress. Exposure of cultured myocytes to 100 micromol/l hydrogen peroxide (H(2)O(2)) increased the number of nuclei stained by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling technique as well as induced internucleosomal DNA fragmentation, loss of mitochondrial membrane potential, cytochrome c release into the cytosol, and activation of caspases-3 and -9, all of which are characteristics of apoptosis. Pretreatment of cells with nicorandil (100 micromol/l) inhibited these effects of H(2)O(2). Both the mitochondrial K(ATP) channel antagonist 5-hydroxydecanoate (5-HD) and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylyl cyclase, attenuated the anti-apoptotic effect of nicorandil in concentration-dependent manners. Coapplication of ODQ (10 micromol/l) and 5-HD (500 micromol/l) completely abolished nicorandil-induced cytoprotection. The effect of nicorandil was also reduced by an inhibitor of cGMP-dependent protein kinase (KT5823, 1 micromol/l). The nitric oxide donor (+/-)-S-nitroso-N-acetylpenicillamine (SNAP, 50 micromol/l) mimicked the protective effect of nicorandil in a manner sensitive to ODQ but not to 5-HD. A cell-permeable cGMP analog, 8-bromo-cGMP, also reduced H(2)O(2)-induced apoptosis. The inhibition of the H(2)O(2)-induced activation of caspase-3, but not that of caspase-9, by nicorandil in the presence of 5-HD or by SNAP was reversed by the addition of dithiothreitol to the enzyme assay. Nicorandil inhibits oxidative stress-induced apoptosis in cardiac myocytes through a nitric oxide/cGMP-dependent mechanism as well as by activating mitochondrial K(ATP) channels.

Paeoniflorin induces apoptosis of lymphocytes through a redox-linked mechanism.

Paeoniflorin (PF), isolated from paeony root, has been used as a herbal medicine for more than 1,200 years in China, Korea, and Japan for its anti-allergic, anti-inflammatory, and immunoregulatory effects. In this study, we found that PF induces apoptosis in both murine T-lineage cells and human T-cell leukemia Jurkat cells. This apoptosis was mediated through the reduction of mitochondrial membrane potential, activation of caspase, and fragmentation of DNA. Interestingly, PF induced generation of reactive oxygen species (ROS) and a reducing agent, dithiothreitol (DTT), and a ROS scavenger, N-acetyl cysteine (NAC), successfully attenuated the PF-induced apoptosis. Additionally, PF induced the phosphorylation of three mitogen-activated protein (MAP) family kinases, extracellular signal-regulated kinase, c-Jun amino-terminal kinase (JNK), and p38 MAP kinase. Curcumin, an anti-oxidant and JNK inhibitor, inhibited PF-induced apoptosis, suggesting the possible involvement of curcumin-sensitive JNK or other redox-sensitive elements in PF-induced apoptosis. These results partially explain the action mechanism of PF-containing paeony root as a herbal medicine.