[Analysis of the mechanism of apoptosis induction by PSK].

Previously, we reported that PSK induces apoptosis and growth inhibition in HL60 cells. In this study, we tried to clarify the mechanism of how PSK induces apoptosis. Because several reports suggested that apoptosis of HL60 cells is mediated by activation of p38MAPK, we examined whether p38MAPK is involved in PSK-induced apoptosis. First, we found that PSK induced p38MAPK phosphorylation, which is considered as its activation. Next, we demonstrated that SB203580, inhibitor of p38MAPK, inhibited PSK-induced apoptosis. These results suggest that p38MAPK plays an important role in PSK-induced apoptosis.

Phenyl sulfate, indoxyl sulfate and p-cresyl sulfate decrease glutathione level to render cells vulnerable to oxidative stress in renal tubular cells.

In chronic kidney disease patients, oxidative stress is generally associated with disease progression and pathogenesis of its comorbidities. Phenyl sulfate is a protein-bound uremic solute, which accumulates in chronic kidney disease patients, but little is known about its nature. Although many reports revealed that protein-bound uremic solutes induce reactive oxygen species production, the effects of these solutes on anti-oxidant level have not been well studied. Therefore, we examined the effects of protein-bound uremic solutes on glutathione levels. As a result, indoxyl sulfate, phenyl sulfate, and p-cresyl sulfate decreased glutathione levels in porcine renal tubular cells. Next we examined whether phenyl sulfate-treated cells becomes vulnerable to oxidative stress. In phenyl sulfate-treated cells, hydrogen peroxide induced higher rates of cell death than in control cells. Buthionine sulfoximine, which is known to decrease glutathione level, well mimicked the effect of phenyl sulfate. Finally, we evaluated a mixture of indoxyl sulfate, phenyl sulfate, and p-cresyl sulfate at concentrations comparable to the serum concentrations of hemodialysis patients, and we confirmed its decreasing effect on glutathione level. In conclusion, indoxyl sulfate, phenyl sulfate, and p-cresyl sulfate decrease glutathione levels, rendering the cells vulnerable to oxidative stress.

Crucial Role of the Aryl Hydrocarbon Receptor (AhR) in Indoxyl Sulfate-Induced Vascular Inflammation.

AIM: The aryl hydrocarbon receptor (AhR), a ligand-inducible transcription factor mediating toxic effects of dioxins and uremic toxins, has recently emerged as a pathophysiological regulator of immune-inflammatory conditions. Indoxyl sulfate, a uremic toxin, is associated with cardiovascular disease in patients with chronic kidney disease and has been shown to be a ligand for AhR. The aim of this study was to investigate the potential role of AhR in indoxyl sulfate-induced leukocyte-endothelial interactions. METHODS: Endothelial cell-specific AhR knockout (eAhR KO) mice were produced by crossing AhR floxed mice with Tie2 Cre mice. Indoxyl sulfate was administered for 2 weeks, followed by injection of TNF-α. Leukocyte recruitment to the femoral artery was assessed by intravital microscopy. Vascular endothelial cells were transfected with siRNA specific to AhR (siAhR) and treated with indoxyl sulfate, followed by stimulation with TNF-α. RESULTS: Indoxyl sulfate dramatically enhanced TNF-α-induced leukocyte recruitment to the vascular wall in control animals but not in eAhR KO mice. In endothelial cells, siAhR significantly reduced indoxyl sulfate-enhanced leukocyte adhesion as well as E-selectin expression, whereas the activation of JNK and nuclear factor-κB was not affected. A luciferase assay revealed that the region between －153 and －146 bps in the E-selectin promoter was responsible for indoxyl sulfate activity via AhR. Mutational analysis of this region revealed that activator protein-1 (AP-1) is responsible for indoxyl sulfate-triggered E-selectin expression via AhR. CONCLUSION: AhR mediates indoxyl sulfate-enhanced leukocyte-endothelial interactions through AP-1 transcriptional activity, which may constitute a new mechanism of vascular inflammation in patients with renal disease.

Classification of Five Uremic Solutes according to Their Effects on Renal Tubular Cells.

Background/Aims. Uremic solutes, which are known to be retained in patients with chronic kidney disease, are considered to have deleterious effects on disease progression. Among these uremic solutes, indoxyl sulfate (IS) has been extensively studied, while other solutes have been studied less to state. We conducted a comparative study to examine the similarities and differences between IS, p-cresyl sulfate (PCS), phenyl sulfate (PhS), hippuric acid (HA), and indoleacetic acid (IAA). Methods. We used LLC-PK1 cells to evaluate the effects of these solutes on viable cell number, cell cycle progression, and cell death. Results. All the solutes reduced viable cell number after 48-hour incubation. N-Acetyl-L-cysteine inhibited this effect induced by all solutes except HA. At the concentration that reduced the cell number to almost 50% of vehicle control, IAA induced apoptosis but not cell cycle delay, whereas other solutes induced delay in cell cycle progression with marginal impact on apoptosis. Phosphorylation of p53 and Chk1 and expression of ATF4 and CHOP genes were detected in IS-, PCS-, and PhS-treated cells, but not in IAA-treated cells. Conclusions. Taken together, the adverse effects of PCS and PhS on renal tubular cells are similar to those of IS, while those of HA and IAA differ.

Protein-bound polysaccharide-K induces apoptosis via mitochondria and p38 mitogen-activated protein kinase-dependent pathways in HL-60 promyelomonocytic leukemia cells.

Protein-bound polysaccharide-K (PSK) is extracted from Coriolus versicolor (CM101). PSK is a biological response modifier (BRM), and its mechanism of action is partly mediated by modulating host immune systems; however, recent studies showed antiproliferative activity of PSK. Therefore, we examined the mechanism underlying the antiproliferative activity of PSK using seven different human malignant cell lines (WiDr, HT29, SW480, KATOIII, AGS, HL-60 and U937), and PSK was found to inhibit the proliferation of HL-60 cells most profoundly. Therefore, HL-60 cells were used to elucidate the mechanism of the antiproliferative activity. Western blotting was performed to detect phosphorylated p38 mitogen-activated protein kinase (MAPK). A p38 MAPK inhibitor, SB203580, was used to examine the roles in PSK-induced apoptosis and growth inhibition. Flow cytometry was performed for mitochondrial membrane potential detection. PSK activated caspase-3 and induced p38 MAPK phosphorylation. Co-treatment with SB203580 blocked PSK-induced apoptosis, caspase-3 activation and growth inhibition. PSK induced apoptosis via the mitochondrial pathway. The depolarization of mitochondria induced by PSK was reversed by co-treatment with SB203580. The present study revealed that PSK induced apoptosis in HL-60 cells via a mitochondrial and p38 MAPK-dependent pathway.

Protein-bound polysaccharide-K augments the anticancer effect of fluoropyrimidine derivatives possibly by lowering dihydropyrimidine dehydrogenase expression in gastrointestinal cancers.

Protein-bound polysaccharide-K (PSK) enhances the antitumor effect of anticancer drug when used clinically in combination with such drugs. PSK is known to act by immune-mediated mechanisms; however, the relationship between PSK and metabolic enzymes of anticancer drugs is unknown. We used the collagen gel droplet-embedded culture drug sensitivity test (CD-DST) clinically to evaluate the sensitivity of anticancer drugs. In the present study, we modified the CD-DST by adding peripheral blood mononuclear cells (PBMCs) (immuno-CD-DST) and examined the antitumor effect of PSK in combination with anticancer drugs. First, HCT116 human colon cancer cells were cultured with PSK and 5-fluorouracil (5-FU) or 5'-deoxy-5-fluorouridine (5'-DFUR) in the presence or absence of PBMCs, and the antiproliferative effects were compared. In the presence of PBMCs, PSK augmented the inhibitory effects of 5-FU and 5'-DFUR on HCT116 cell proliferation. Next, using human gastric cancer and colon cancer cell lines, the effects of PSK on mRNA expression of various metabolic enzymes of fluoropyrimidines: dihydropyrimidine dehydrogenase (DPD), thymidylate synthase, thymidine phosphorylase and orotate phosphoribosyl transferase, were examined by real-time PCR. PSK significantly enhanced DPD mRNA expression in all of the cancer cell lines tested, but not those of the other enzymes. Addition of IFN-α and TRAIL, cytokines known to inhibit DPD expression, to the cultures reduced DPD mRNA expression in the cancer cells. When PBMC samples collected from healthy volunteers were cultured with PSK, IFN-α mRNA expression increased in 3 of the 5 PBMC samples, while TRAIL mRNA expression was unchanged. The present results propose the possibility that PSK induces PBMCs to express IFN-α which inhibits DPD expression, and consequently augments the antitumor effect of 5-FU or 5'-DFUR. Immuno-CD-DST is useful for evaluating drugs with immunological mechanisms of action.

Protein-bound polysaccharide-K (PSK) induces apoptosis via p38 mitogen-activated protein kinase pathway in promyelomonocytic leukemia HL-60 cells.

BACKGROUND/AIM: Protein-bound polysaccharide-K (PSK) is extracted from Coriolus versicolor (CM101) and is clinically used in combination therapy for gastrointestinal cancer and small-cell lung carcinoma. We have previously demonstrated that PSK induces apoptosis and inhibites proliferation of promyelomonocytic leukemia HL-60 cells, but the signaling pathway for this action remains to be elucidated. In HL-60 cells, the mitogen-activated protein kinase (MAPK) pathway has been reported to be involved in stimuli-induced apoptosis. Therefore, involvement of the p38 MAPK pathway in PSK-induced apoptosis was herein investigated. MATERIALS AND METHODS: HL-60 cells were used in this study. Western blotting was performed to detect phosphorylated p38 MAPK. A p38 MAPK inhibitor, SB203580, was used to examine the roles of p38 MAPK in PSK-induced apoptosis and growth inhibition. RESULTS: PSK induced p38 MAPK phosphorylation. Co-treatment with SB203580 blocked PSK-induced apoptosis, caspase-3 activation and growth inhibition. CONCLUSION: The p38 MAPK pathway plays an important role in PSK-induced apoptosis.

Protein-bound polysaccharide-K (PSK) induces apoptosis and inhibits proliferation of promyelomonocytic leukemia HL-60 cells.

Protein-bound polysaccharide-K (PSK) is extracted from Coriolus versicolor (CM101), and is clinically used in combination therapy for gastrointestinal cancer and small cell lung carcinoma. PSK is a biological response modifier (BRM), and its mechanism of action is partly mediated, by modulating host immune systems, such as the activation of immune effector cells and the neutralization of transforming growth factor-beta (TGFß) activity. Direct inhibition of tumor cell proliferation has been reported as another mechanism, but how PSK induces such an effect remains to be elucidated. Here, the anti-proliferative activity of PSK was examined using seven different human malignant cell lines (WiDr, HT29, SW480, KATOIII, AGS, HL60 and U937), and PSK was found to inhibit the proliferation of HL-60 cells most profoundly. Therefore, HL-60 cells were used to clarify the mechanism of anti-proliferative activity. Caspase-3 activation followed by apoptosis are involved at least in part in the PSK-induced anti-proliferative activity against HL-60 cells.

Brugia malayi asparaginyl-transfer RNA synthetase induces chemotaxis of human leukocytes and activates G-protein-coupled receptors CXCR1 and CXCR2.

Background. Lymphatic filariasis is a chronic human parasitic disease in which the parasites repeatedly provoke acute and chronic inflammatory reactions in the host bloodstream and lymphatics. Excretory-secretory products derived from filariae are believed to play an important role in the development of associated immunologic conditions; however, the specific mechanisms involved in these changes are not well understood. Recently, human cytoplasmic aminoacyl-transfer (t) RNA synthetases, which are autoantigens in idiopathic inflammatory myopathies, were shown to activate chemokine receptors on T lymphocytes, monocytes, and immature dendritic cells by recruiting immune cells that could induce innate and adaptive immune responses. Filarial (Brugia malayi) asparaginyl-tRNA synthetase (AsnRS) is known to be an immunodominant antigen that induces strong human immunoglobulin G3 responses.Methods. Recombinant B. malayi AsnRS was used to perform cellular function assays--for example, chemotaxis and kinase activation assays.Results. Unlike human AsnRS, parasite AsnRS is chemotactic for neutrophils and eosinophils. Recombinant B. malayi AsnRS but not recombinant human AsnRS induced chemotaxis of CXCR1 and CXCR2 single-receptor-transfected HEK-293 cell lines, blocked CXCL1-induced calcium flux, and induced mitogen-activated protein kinase.Conclusions. Our findings suggest that a filarial parasite chemoattractant protein may contribute to the development of chronic inflammatory disease and that chemokine receptors may be therapeutic targets to ameliorate parasite-induced pathology.

A duodenally absorbable CXC chemokine receptor 4 antagonist, KRH-1636, exhibits a potent and selective anti-HIV-1 activity.

A low molecular weight nonpeptide compound, KRH-1636, efficiently blocked replication of various T cell line-tropic (X4) HIV type 1 (HIV-1) in MT-4 cells and peripheral blood mononuclear cells through the inhibition of viral entry and membrane fusion via the CXC chemokine receptor (CXCR)4 coreceptor but not via CC chemokine receptor 5. It also inhibited binding of the CXC chemokine, stromal cell-derived factor 1alpha, to CXCR4 specifically and subsequent signal transduction. KRH-1636 prevented monoclonal antibodies from binding to CXCR4 without down-modulation of the coreceptor. The inhibitory effect against X4 viral replication by KRH-1636 was clearly reproduced in the human peripheral blood lymphocytesevere combined immunodeficiency mouse system. Furthermore, this compound was absorbed into the blood after intraduodenal administration as judged by anti-HIV-1 activity and liquid chromatography MS in the plasma. Thus, KRH-1636 seems to be a promising agent for the treatment of HIV-1 infection.