Selectively induced apoptosis in human neutrophils in the presence of oxidative phenoxazines, 2-amino-4,4α-dihydryo-4α-7H-phenoxazine-3-one and 2-aminophenoxazine-3-one, preceded by decrease of intracellular pH, depolarization of the mitochondria, and inhibition of superoxide generation.

The present research investigated the effect of the oxidative phenoxazines, 2-amino-4,4α-dihydryo-4α-7H-phenoxazine-3-one (Phx-1) and 2-amino-phenoxazine-3-one (Phx-3) on apoptosis induction and apoptosis-related early events in human neutrophils. When Phx-1 or Phx-3 was administered to freshly drawn human blood for 18 h, these phenoxazines caused apoptotic cell death morphologically characterized by condensation of the nucleus in neutrophils, without causing it in lymphocytes and monocytes. Apoptosis, which was detectable by microscopic analysis and by using flow-cytometry, occurred significantly in human neutrophils isolated from freshly drawn blood, 6 h after the administration of 50 µM Phx-1 and Phx-3. After 24 h, every isolated neutrophil treated with Phx-1 or Phx-3 fell into apoptosis or lost its morphology, while many of the neutrophils without these phenoxazines remained alive, with normal morphology. Apoptosis-related early events including a decrease in intracellular pH (pHi) and depolarization of the mitochondria occurred in the isolated neutrophils, 30 min and 6 h after the administration of Phx-1 or Phx-3, respectively. Superoxide generation from the isolated neutrophils mimicked by phorbol myristate acetate (PMA) was very markedly inhibited by 100 µM Phx-1 or Phx-3. This result could be explained, in part, by the fact that the insufficient supply of NADPH (nicotinamide adenine dinucleotide phosphate, reduced form) was caused by pHi decrease in neutrophils treated with Phx-1 or Phx, because NADPH is necessary for NADPH oxidase responsible for generating superoxide in the cells. The present results suggest that Phx-1 and Phx-3 have the capacity of selectively inducing apoptosis in human neutrophils and that these phenoxazines may be useful as specific drugs to induce apoptotic cell death of human neutrophils and thereby prevent inflammation caused by these phagocytic cells.

MK615 decreases RAGE expression and inhibits TAGE-induced proliferation in hepatocellular carcinoma cells.

AIM: To investigate the proliferative effect of advanced glycation end-products (AGEs) and the role of their cellular receptor (RAGE) on hepatocellular carcinoma (HCC) cells, and the inhibitory effects of MK615, an extract from Japanese apricot, against AGEs were also evaluated. METHODS: Two HCC cell lines, HuH7 and HepG2, were used. Expression of RAGE was investigated by polymerase chain reaction, Western blotting, and flow cytometry (FACS). The effect of MK615 on RAGE expression was also evaluated by FACS. The proliferative effects of a control (unglycated bovine serum albumin), glucose-derived AGEs (Glc-AGE), and glyceraldehyde-derived AGEs (Glycer-AGE), and the anti-proliferative effect of MK615 against AGEs, were evaluated using MTT assays. RESULTS: Expression of RAGE was confirmed at both the mRNA and protein levels in both HuH7 and HepG2. FACS revealed that the level of RAGE expression was higher in HuH7 than in HepG2. Treatment with 0.1 µg/mL MK615 decreased the expression level of RAGE from 24.3% to 3.7% in HuH7 and from 6.2% to 4.8% in HepG2. The growth indices for the control, Glc-AGE, and Glycer-AGE were 1.06 ± 0.08, 0.99 ± 0.04, and 1.38 ± 0.05, respectively, in HuH7 (P = 0.037), and were 1.03 ± 0.04, 1.04 ± 0.03, and 1.07 ± 0.05, respectively, in HepG2 (P > 0.05). When the cells were cultured simultaneously with Glycer-AGE and MK615, MK615 abrogated the proliferative effect of Glycer-AGE in HuH7. CONCLUSION: Only Glycer-AGE has a proliferative effect on HuH7, which expresses a higher level of RAGE. MK615 suppresses the proliferative effect of Glycer-AGE on HuH7 by decreasing the expression of RAGE.

Mechanism of HMGB1 release inhibition from RAW264.7 cells by oleanolic acid in Prunus mume Sieb. et Zucc.

High mobility group box-1 protein (HMGB1), primarily from the nucleus, is released into the extracellular milieu either passively from necrotic cells or actively through secretion by monocytes/macrophages. Extracellular HMGB1 acts as a potent inflammatory agent by promoting the release of cytokines such as tumor necrosis factor (TNF)-alpha, has procoagulant activity, and is involved in death due to sepsis. Accordingly, HMGB1 is an appropriate therapeutic target. In this study, we found that an extract of Prunus mume Sieb. et Zucc. (Ume) fruit (Ume extract), an abundant source of triterpenoids, strongly inhibited HMGB1 release from lipopolysaccharide (LPS)-stimulated macrophage-like RAW264.7 cells. The inhibitory effect on HMGB1 release was enhanced by authentic oleanolic acid (OA), a naturally occurring triterpenoid. Similarly, the HMGB1 release inhibitor in Ume extract was found to be OA. Regarding the mechanisms of the inhibition of HMGB1 release, the OA or Ume extract was found to activate the transcription factor Nrf2, which binds to the antioxidative responsive element, and subsequently the heme oxygenase (HO)-1 protein was induced, indicating that the inhibition of HMGB1 release from LPS-stimulated RAW264.7 cells was mediated via the Nrf2/HO-1 system; an essentially antioxidant effect. These results suggested that natural sources of triterpenoids warrant further evaluation as 'rescue' therapeutics for sepsis and other potentially fatal systemic inflammatory disorders.

MK615 inhibits pancreatic cancer cell growth by dual inhibition of Aurora A and B kinases.

AIM: To investigate the anti-neoplastic effect of MK615, an anti-neoplastic compound isolated from Japanese apricot, against human pancreatic cancer cells in vitro. METHODS: Three human pancreatic cancer cell lines PANC-1, PK-1, and PK45H were cultured with MK615 at concentrations of 600, 300, 150, and 0 microg/mL. Growth inhibition was evaluated by cell proliferation assay, and killing activity was determined by lactate dehydrogenase (LDH) assay. Expression of Aurora A and B kinases was detected by real-time polymerase chain reaction (PCR) and Western blotting. Cell cycle stages were evaluated by flow cytometry. RESULTS: The growth inhibitory rates of MK615 at 150, 300, and 600 microg/mL were 2.3%+/-0.9%, 8.9%+/-3.2% and 67.1%+/-8.1% on PANC1 cells, 1.3%+/-0.3%, 8.7%+/-4.1% and 45.7+/-7.6% on PK1 cells, and 1.2+/-0.8%, 9.1%+/-2.1% and 52.1%+/-5.5% on PK45H cells, respectively (P<0.05). The percentage cytotoxicities of MK615 at 0, 150, 300, and 600 microg/mL were 19.6%+/-1.3%, 26.7%+/-1.8%, 25.5%+/-0.9% and 26.4%+/-0.9% in PANC1 cells, 19.7%+/-1.3%, 24.7%+/-0.8%, 25.9%+/-0.9% and 29.9%+/-1.1% in PK1 cells, and 28.0%+/-0.9%, 31.2%+/-0.9%, 30.4%+/-1.1% and 35.3+/-1.0% in PK45H cells, respectively (P<0.05). Real-time PCR and Western blotting showed that MK615 dually inhibited the expression of Aurora A and B kinases. Cell cycle analysis revealed that MK615 increased the population of cells in G2/M phase. CONCLUSION: MK615 exerts an anti-neoplastic effect on human pancreatic cancer cells in vitro by dual inhibition of Aurora A and B kinases.

New anti-proliferative agent, MK615, from Japanese apricot "Prunus mume" induces striking autophagy in colon cancer cells in vitro.

AIM: To investigate the anti-neoplastic effects of MK615, an extract from the Japanese apricot (Prunus mume), against colon cancer cells. METHODS: Three colon cancer cell lines, SW480, COLO, and WiDr, were cultured with MK615. Growth inhibition was evaluated by cell proliferation assay and killing activity was determined by lactate dehydrogenase assay. Induction of apoptosis was evaluated by annexin V flow cytometry. Morphological changes were studied by light and electron microscopy, and immunofluorescence staining with Atg8. RESULTS: MK615 inhibited growth and lysed SW480, COLO and WiDr cells in a dose-dependent manner. Annexin V flow cytometry showed that MK615 induced apoptosis after 6 h incubation, at which point the occurrence of apoptotic cells was 68.0%, 65.7% and 64.7% for SW480, COLO, and WiDr cells, respectively. Light and electron microscopy, and immunofluorescence staining with Atg8 revealed that MK615 induced massive cytoplasmic vacuoles (autophagosomes) in all three cell lines. CONCLUSION: MK615 has an anti-neoplastic effect against colon cancer cells. The effect may be exerted by induction of apoptosis and autophagy.

A novel anti-cancer substance, MK615, from ume, a variety of Japanese apricot, inhibits growth of hepatocellular carcinoma cells by suppressing Aurora A kinase activity.

BACKGROUND/AIMS: MK615 is an anti-cancer substance extracted from the Japanese apricot. In the present study, the anti-neoplastic effect of MK615 against hepatocellular carcinoma (HCC) was evaluated in vitro, and its mechanism was elucidated. METHODOLOGY: Two HCC lines, HuH7 and Hep3B, were cultured with MK615 at concentrations of 600, 300, 150, and 0 microg/mL. Growth inhibition was evaluated by MTT assay, and killing activity was determined by LDH assay. Cell cycle stages were evaluated by flow cytometry. Expression of Aurora A kinase (Aurora A) was evaluated by real-time PCR and Western blotting, and inhibition of Aurora A activity was determined by HTscan. RESULTS: MK615 inhibited the growth of, and lysed, HuH7 and Hep3B cells in a dose-dependent manner. Cell cycle analysis revealed that MK615 increased the population of cells in G2/M phase. Real-time PCR and Western blotting showed that MK615 suppressed the expression of Aurora A. HTscan assay demonstrated that Aurora A activity was specifically inhibited by 34.3%, 32.9%, and 54.3% at 150, 300, and 600 microg/mL MK615, respectively. CONCLUSIONS: MK615 has an anti-cancer effect against HCC lines in vitro, and the effect is exerted through inhibition of Aurora A activity.

New antineoplastic agent, MK615, from UME (a Variety of) Japanese apricot inhibits growth of breast cancer cells in vitro.

MK615 is an extract mixture containing hydrophobic substances from Japanese apricot. In this study, the antineoplastic effects of MK615 against breast cancer cells were investigated. Two breast cancer cell lines, MDA-MB-468 (MDA) and MCF7, were cultured with (600, 300, and 150 mug/mL) or without MK615. After 72 hours of incubation, growth inhibition was evaluated by MTT assay. The cells were then cultured with MK615 (300 mug/mL) and morphological changes were studied by light and electron microscopy. Finally, the mechanism of the antineoplastic effect of MK615 was evaluated by cell cycle and apoptosis assay. MK615 inhibited the growth of MDA and MCF7 in a dose-dependent manner. The percentage growth inhibition of MDA at dosages of 600, 300, and 150 mug/mL was 59.2%, 52.4%, and 23.3%, respectively, and that for MCF7 was 83.5%, 52.7%, and 16.6%, respectively. Morphological changes after MK615 treatment included massive vacuolization in the cytoplasm and apoptotic changes in the nucleus. These changes began to be apparent after at least 6 hours of incubation. Cell cycle analysis showed that MK615 increased the proportion of cells in the G2-M phase in both MDA (7.8-11.7%) and MCF7 (8.1-18.7%), and finally both cell lines became apoptotic. The proportion of apoptotic cells increased with incubation time. MK615 effectively inhibits the growth of breast cancer cells in vitro, possibly by cell cycle modification and apotosis induction. MK615 should be further investigated as a promising anti-breast cancer agent.

Induction of HIV-specific antibody response and protection against vaginal SHIV transmission by intranasal immunization with inactivated SHIV-capturing nanospheres in macaques.

We have previously reported that concanavalin A-immobilized polystyrene nanospheres (Con A-NS) could efficiently capture HIV-1 particles and that intranasal immunization with inactivated HIV-1-capturing nanospheres (HIV-NS) induced vaginal anti-HIV-1 IgA antibody response in mice. In this study, to evaluate the protective effect of immunization, each three macaques was intranasally immunized with Con A-NS or inactivated simian/human immunodeficiency virus KU-2-capturing nanospheres (SHIV-NS) and then intravaginally challenged with a pathogenic virus, SHIV KU-2. After a series of six immunizations, vaginal anti-HIV-1 gp120 IgA and IgG antibodies were detected in all SHIV-NS-immunized macaques. After intravaginal challenge, one of the three macaques in each of the Con A-NS- and SHIV-NS-immunized groups was infected. Plasma viral RNA load of infected macaque in SHIV-NS-immunized macaques was substantially less than that in unimmunized control macaque and reached below the detectable level. However, it could not be determined whether intranasal immunization with SHIV-NS is effective in giving complete protection against intravaginal challenge. To explore the effect of the SHIV-NS vaccine, the remaining non-infected macaques were rechallenged intravenously with SHIV KU-2. After intravenous challenge, all macaques became infected. However, SHIV-NS-immunized macaques had lower viral RNA loads and higher CD4(+) T cell counts than unimmunized control macaques. Plasma anti-HIV-1 gp120 IgA and IgG antibodies were induced more rapidly in the SHIV-NS-immunized macaques than in the controls. The rapid antibody responses having neutralizing activity might contribute to the clearance of the challenge virus. Thus, SHIV-NS-immunized macaques exhibited partial protection to vaginal and systemic challenges with SHIV KU-2.

Validity of plasma remnant lipoproteins as surrogate markers of antemortem level in cases of sudden coronary death.

Hitherto triglycerides (TG) and TG-rich lipoproteins were been of limited value as surrogates for antemortem levels. We measured TG levels in postmortem plasma from sudden coronary death cases (SCD, n=91) by using two TG assays, Dry Chem TG (free glycerol was added) and the Determiner L-TG (without added free glycerol) that measured net TG. TG levels were markedly higher by the Dry Chem TG (y) vs. Determiner L-TG (x), y = 1.03x + 229 mg/dl. HPLC showed large amounts of free glycerol in postmortem plasma and in TG-rich lipoprotein remnants (RLP). These results were verified in a rabbit model of SCD. Further, RLP from SCD were found to be biophysically similar to those from living patients with coronary artery disease (CAD). In conclusion, postmortem plasma sampled up to 12 h after death is appropriate for measuring lipid and lipoproteins, TG and RLP-TG as surrogates for antemortem levels when a TG assay without added free glycerol is used.

Glycosylphosphatidyl inositol-anchored protein (GPI-80) gene expression is correlated with human thymoma stage.

Thymoma is one of the most common solid tumors in the mediastinum. Because there is no typical cell line for human thymoma, the development and use of molecular-based therapy for thymoma will require detailed molecular-genetic analysis of patients' tissues. Recent reports showed that genetic aberrations in thymoma were most frequently seen in chromosome 6q regions. We investigated the use of oligonucleotide arrays to monitor in vivo expression levels of genes in chromosome 6 regions in early- (stage I or II) and late- (stage IVa) stage thymoma tissues from patients. These in vivo gene expression profiles were verified by real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) using LightCycler for 48 thymoma patients and sandwich ELISA for 33 thymoma patients. Using both methods, a candidate gene was identified which was overexpressed in stage IV thymoma. This was a known glycosylphosphatidyl inositol (GPI)-anchored protein (GPI-80), which is highly homologous with Vanin-1, a mouse thymus homing protein. Serum level of GPI-80 was confirmed to be elevated in stage IV thymoma compared with in stage I thymoma by using sandwich ELISA. The combined use of oligonucleotide microarray, real-time RT-PCR, and ELISA analyses provides a powerful new approach to elucidate the in vivo molecular events surrounding the development and progression of thymoma.

Studies on the mechanisms of leukocyte adhesion to cellulose acetate beads: an in vitro model to assess the efficacy of cellulose acetate carrier-based granulocyte and monocyte adsorptive apheresis.

Granulocyte and monocyte adsorptive apheresis (GMA) using a column filled with cellulose acetate (CA) beads (carriers) has been associated with a significant clinical efficacy in patients with rheumatoid arthritis and ulcerative colitis. To obtain further understanding on the mechanisms of disease modification by cellulose acetate-carrier-based GMA, in the present study, we investigated the mechanisms of granulocyte and monocyte adhesion to CA beads following exposure of human peripheral blood to the carriers at 37 degrees C for up to 60 min under controlled conditions. Cellulose acetate beads selectively adsorbed granulocytes, monocytes. CD19+ (B cells) and CD56+ (NK cells) lymphocyte subpopulations. The granulocyte and monocyte adsorption was inhibited by heat-inactivated plasma and EDTA, indicating that the adsorption was plasma protein (immunoglobulin, complement) and calcium dependent. Accordingly, granulocyte and monocyte adsorption was markedly enhanced by coating the carriers with IgG. Similarly, C3b was adsorbed onto the CA beads as a marker of complement activation. The results indicated that IgG and active complement fragments mediated leukocyte adhesion to CA beads via the FcgammaR and/or leukocyte complement receptor like CR3. Additionally, CA beads induced loss of expression of TNF receptors on CD16- granulocytes and CD14+ monocytes, but not on CD3+ lymphocytes In conclusion, CA beads might be an appropriate biomaterial for inducing extracorporeal immunomodulation as a treatment for auto-immune diseases which are associated with pathological leukocyte activity.

Mucosal immunization with inactivated HIV-1-capturing nanospheres induces a significant HIV-1-specific vaginal antibody response in mice.

Mucosal secretory IgA is considered to have an important role in the prevention of human immunodeficiency virus type 1 (HIV-1) transmission through sexual intercourse. Therefore, substances that induce HIV-1-specific IgA antibody in the genital tract may become promising candidates for prophylactic vaccine against HIV-1 infection. We have previously reported that concanavalin A-immobilized polystyrene nanospheres (Con A-NS) could efficiently capture HIV-1 particles and gp120 antigens on their surface and that intravaginal immunization with inactivated HIV-1-capturing nanospheres (HIV-NS) induced vaginal anti-HIV-1 IgA antibody in mice. In this study, various strategies for immunization with HIV-NS were undertaken to induce HIV-1-specific IgA response in the mouse genital tract. HIV-NS were administered intravaginally, orally, intranasally or intraperitoneally to mice. Progesterone treatment enhanced the anti-HIV-1 IgA response to intravaginal immunization significantly, but intranasal immunization with HIV-NS was more effective compared with other immunization routes in terms of vaginal IgA response. In addition, vaginal washes from intranasally immunized mice were capable of neutralizing HIV-1(IIIB). Thus, application of HIV-NS is a practical approach to promote HIV-1-specific IgA response by the vaginal mucosa in the mouse and intranasal appears to be an effective immunization route in this animal model. Intranasal immunization with HIV-NS should be further pursued for its potential as an HIV-1 prophylactic vaccine.

Mechanism of superoxide anion production by hepatic sinusoidal endothelial cells and Kupffer cells during short-term ethanol perfusion in the rat.

BACKGROUND/AIMS: The aim of this study was to clarify the candidate cells for and the mechanism of superoxide anion (O2*-) release into the hepatic sinusoids during short-term exposure to ethanol. METHODS: The rat liver was perfused continuously with ethanol (a substrate for alcohol dehydrogenase) or tert-buthanol (not a substrate for alcohol dehydrogenase) for 20 min at a final concentration of 40 mM. In order to detect O2*- production, MCLA (2-methyl-6-[p-methoxyphenyl]-3,7-dihydroimidazo[1,2-a]pyrazin-3-one), a Cypridina luciferin analogue, was simultaneously infused and MCLA-enhanced chemiluminescence was measured. The effects of gadolinium chloride (GdCL3) (a suppressor of Kupffer cells (KCs)), staurosporine (ST) (an inhibitor of serine-threonine kinases, including protein kinase C), diphenyleneiodonium chloride (DPI) (an inhibitor of NADPH oxidase), ibuprofen (IB) (an inhibitor of cyclooxygenase) and 4-methylpyrazole (4MP) (an inhibitor of ethanol metabolism) on the ethanol-induced chemiluminescence were also evaluated. Sites where O2*- could be released were determined by histochemical detection of nitro blue tetrazolium reduction. RESULTS: Both ethanol and tert-buthanol rapidly caused O2*- release. GdCL3 suppressed the ethanol-induced O2*- release by 61%. Staurosporine and DPI, but neither IB nor 4-MP, also significantly inhibited the ethanol-induced O2*- release. In the histochemical examination, ethanol-stimulated liver showed blue formazan precipitate on both sinusoidal endothelial cells (SECs) and Kupffer cells (KCs), whereas the GdCl3-pretreated liver had the precipitate only on SECs. CONCLUSIONS: This study shows that ethanol itself stimulates both SECs and KCs to release O2*- via activation of NADPH oxidase probably involving protein kinase C (PKC).

Anti-inflammatory effect of granulocyte and monocyte adsorption apheresis in a rabbit model of immune arthritis.

In active rheumatoid arthritis, large numbers of granulocytes and macrophages are found in the inflamed joints. These leucocytes can promote inflammation and tissue injury by releasing inflammatory cytokines, proteinases and oxygen derivatives. To see if granulocyte and monocyte (GM) depletion produces anti-inflammatory effect, GM adsorption apheresis was performed in rabbits with immune arthritis by using a column (Adacolumn) filled with cellulose diacetate beads (G-1 beads) as adsorptive carriers which selectively adsorb CD11b positive GMs. Injection of ovalbumin into the knee joints of ovalbumin-sensitized rabbits caused a marked increase in peripheral blood leucocytes, joint swelling, increased granulocyte adhesion to G-1 beads and elevated TNF-alpha production by peripheral blood mononuclear cells (PBMC). When rabbits received a 60 min adsorption apheresis, there was suppression of CD11b positive leucocyte infiltration into the joint and reduced joint swelling (P < 0.01) compared with controls. Additionally, there was a significant (p < 0.01) suppression of TNF-alpha production by PBMC in the post column blood. These results suggest that GM depletion may serve as a non-pharmacological strategy to modify inflammatory disorders.

Immunomodulatory effects of granulocyte and monocyte adsorption apheresis as a treatment for patients with ulcerative colitis.

Our aim was to understand the mechanism of immunological changes associated with the use of an adsorptive-type extracorporeal device (Adacolumn) that has been developed for selective adsorption of granulocytes and monocytes/macrophages from peripheral blood of patients with active ulcerative colitis. The column is filled with carriers (G-1 beads) that have a diameter of 2 mm and are made of cellulose diacetate. In peripheral blood treated with the G-1 beads or peripheral blood from patients with active ulcerative colitis following granulocyte and monocyte adsorption apheresis, a significant suppression of proinflammatory cytokines (tissue necrosis factor-alpha, interleukin-1beta, interleukin-6, and interleukin-8) production by leukocytes, neutrophil chemotaxis, down-regulation of leukocyte adhesion molecule (L-selectin) and neutrophil adhesion to interleukin-1beta-activated endothelial cells were observed. Furthermore, after granulocyte adsorption therapy, the number of CD10-negative premature granulocytes increased, indicating increased turnover of these cells in the circulation. Our observations suggest that selective granulocyte and monocyte adsorption is associated with modified peripheral blood leukocyte function favorable to patients with ulcerative colitis and possibly other autoimmune disorders which reflect leukocyte hyperactivity.