Inflammation-related signaling pathways implicating TGFß are revealed in the expression profiling of MCF7 cell treated with fermented soybean, chungkookjang.

Chungkookjang is a Korean fermented soybean containing microorganisms, proteinase, and diverse bioactive compounds, including a high concentration of isoflavones and peptides. Growth of breast cancer MCF7 cells decreased dependent on the concentration of fermented soybean extracts. The effect of fermented soybean on cellular gene expression was determined in a systematic manner comprehensively. DNA microarray analysis was performed using 25,804 probes. Ninety one genes whose expression levels were significantly changed were selected. TGFßI and Smad3 were upregulated. Downregulation of inflammation-related CSF2, CSF2RA, and CSF3 was found. Differential expression of chemokines CCL2, CCL3, CCL3L3, CXCL1, and CXCL2 were observed. Network analysis identified ERß in the network. Based on the experimental results, taking fermented soybean might be helpful for preventing breast cancer by a mechanism activating TGFß pathway and depressing inflammation.

Ethanol extract of fermented soybean, Chungkookjang, inhibits the apoptosis of mouse spleen, and thymus cells.

Apoptosis is a step of the cell cycle which is important in the regulation of immune cell populations. Chungkookjang is a Korean traditional fermented soybean containing microorganisms, enzymes, and bioactive compounds which was used in the treatment of mouse spleen as well as thymus cells (CH1-fermented soybean containing barley, wormwood, and sea tangle; CH2-fermented soybean) and was found to exhibit substantially reduced small DNA fragmentation. An MTT assay showed that the treatment of CH1 and CH2 into the mouse splenocytes and thymocytes sharply increased their survival. Moreover, a FACS analysis also showed that CH1 and CH2 are effective at suppressing the apoptosis of splenocytes and thymocytes. The fermented soybean isoflavone concentrations, which are implicated in lowering breast and prostate cancers, lowering the risk of cardiovascular diseases, and improving bone health, were determined using Capillary Electrophoresis-Electrochemical Detection (CE-ED). The amount of Daidzein in fermented soybean significantly increased by 44-fold dramatically, compared with those in unfermented soybean. In this study, we demonstrated that ethanol extracts of Chungkookjang promote the survival of the mouse spleen and thymus cells in culture by suppressing their apoptotic death. Future studies should investigate which genes are related to apoptosis of the immune cells.

Specific oligopeptides in fermented soybean extract inhibit NF-κB-dependent iNOS and cytokine induction by toll-like receptor ligands.

The ethanol extract of fermented soybean from Glycine max (chungkookjang, CHU) has been claimed to have chemopreventive and cytoprotective effects. In the present study, we examined the inhibitory effect of CHU on inducible nitric oxide synthase (iNOS) and cytokine induction by toll-like receptor (TLR) ligands treatment and attempted to identify the responsible active components. Nitric oxide (NO) content and iNOS levels in the media or RAW264.7 cells were measured using the Griess reagent and real-time polymerase chain reaction assays. CHU treatment inhibited NO production and iNOS induction elicited by lipopolysaccharide (LPS, TLR4L) in a concentration-dependent manner. Tumor necrosis factor-α and interleukin-6 productions were also diminished. Peptidoglycans (TLR2/6L) and CpG-oligodeoxynucleotides (TLR9L) from CHU inhibited iNOS induction, but not poly I:C (TLR3L) or loxoribine (TLF7L). The anti-inflammatory effect resulted from the inhibition of nuclear factor-kappa B (NF-κB) through the inhibition of inhibitory-κB degradation. Of the representative components in CHU, specific oligopeptides (AFPG and GVAWWMY) had the ability to inhibit iNOS induction by LPS, whereas others failed to do so. Daidzein, an isoflavone used for comparative purposes, was active at a relatively higher concentration. In an animal model, oral administration of CHU to rats significantly diminished carrageenan-induced paw edema and iNOS induction. Our results demonstrate that CHU has anti-inflammatory effects against TLR ligands by inhibiting NF-κB activation, which may result from specific oligopeptide components in CHU. Since CHU is orally effective, dietary applications of CHU and/or the identified oligopeptides may be of use in the prevention of inflammatory diseases.

Protein interaction network related to Helicobacter pylori infection response.

AIM: To understand the complex reaction of gastric inflammation induced by Helicobacter pylori (H pylori) in a systematic manner using a protein interaction network. METHODS: The expression of genes significantly changed on microarray during H pylori infection was scanned from the web literary database and translated into proteins. A network of protein interactions was constructed by searching the primary interactions of selected proteins. The constructed network was mathematically analyzed and its biological function was examined. In addition, the nodes on the network were checked to determine if they had any further functional importance or relation to other proteins by extending them. RESULTS: The scale-free network showing the relationship between inflammation and carcinogenesis was constructed. Mathematical analysis showed hub and bottleneck proteins, and these proteins were mostly related to immune response. The network contained pathways and proteins related to H pylori infection, such as the JAK-STAT pathway triggered by interleukins. Activation of nuclear factor (NF)-kappaB, TLR4, and other proteins known to function as core proteins of immune response were also found. These immune-related proteins interacted on the network with pathways and proteins related to the cell cycle, cell maintenance and proliferation, and transcription regulators such as BRCA1, FOS, REL, and zinc finger proteins. The extension of nodes showed interactions of the immune proteins with cancer-related proteins. One extended network, the core network, a summarized form of the extended network, and cell pathway model were constructed. CONCLUSION: Immune-related proteins activated by H pylori infection interact with proto-oncogene proteins. The hub and bottleneck proteins are potential drug targets for gastric inflammation and cancer.

Anti-apoptotic activity of laminarin polysaccharides and their enzymatically hydrolyzed oligosaccharides from Laminaria japonica.

Laminarin polysaccharides (LP1) were prepared from Laminaria japonica, a marine brown alga with potential biological activities, by hot water extraction, ultrafiltration and gel chromatography; the molecular weights of the LP1s were between 5 and 10 kDa. Laminarin oligosaccharides (LO) derived by hydrolyzing LP1 with an endo-beta-(1-->3)-glucanase from Bacillus circulans were mainly di- and penta-oligosaccharides. Treatment of mouse thymocytes with LO or LP1 (1-4 mg ml(-1)) suppressed apoptotic death around 3- or 2-fold and extended cell survival in culture at a rate of about 30 or 20%. A mouse cDNA microarray showing the genes coding for immune response proteins were induced and apoptotic cell death proteins were reduced significantly by LO provided preliminary information regarding the immunomodulatory mechanism of LO. These results suggest that laminarin oligosaccharides and polysaccharides can be utilized to develop new immunopotentiating substances and functional alternative medicines.

Vibrio harveyi nitroreductase is also a chromate reductase.

The chromate reductase purified from Pseudomonas ambigua was found to be homologous with several nitroreductases. Escherichia coli DH5alpha and Vibrio harveyi KCTC 2720 nitroreductases were chosen for the present study, and their chromate-reducing activities were determined. A fusion between glutathione S-transferase (GST) and E. coli DH5alpha NfsA (GST-EcNfsA), a fusion between GST and E. coli DH5alpha NfsB (GST-EcNfsB), and a fusion between GST and V. harveyi KCTC 2720 NfsA (GST-VhNfsA) were prepared for their overproduction and easy purification. GST-EcNfsA, GST-EcNFsB, and GST-VhNFsA efficiently reduced nitrofurazone and 2,4,6-trinitrotoluene (TNT) as their nitro substrates. The K(m) values for GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA for chromate reduction were 11.8, 23.5, and 5.4 micro M, respectively. The V(max) values for GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA were 3.8, 3.9, and 10.7 nmol/min/mg of protein, respectively. GST-VhNfsA was the most effective of the three chromate reductases, as determined by each V(max)/K(m) value. The optimal temperatures of GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA for chromate reduction were 55, 30, and 30 degrees C, respectively. Thus, it is confirmed that nitroreductase can also act as a chromate reductase. Nitroreductases may be used in chromate remediation. GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA have a molecular mass of 50 kDa and exist as a monomer in solution. Thin-layer chromatography showed that GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA contain FMN as a cofactor. GST-VhNfsA reduced Cr(VI) to Cr(III). Cr(III) was much less toxic to E. coli than Cr(VI).

Induction of apoptosis by 3,4'-dimethoxy-5-hydroxystilbene in human promyeloid leukemic HL-60 cells.

3, 4'-Dimethoxy-5-hydroxystilbene (DMHS) is a hydroxystilbene compound obtained by methylation and acid hydrolysis of piceid (resveratrol-3-O-glucoside) from Polygonum cuspidatum. Herein, we report that DMHS induces programmed cell death or apoptosis in human promyelocytic leukemic HL-60 cells. We found that treatment of HL-60 cells with DMHS suppressed the cell growth in a concentration-dependent manner with an IC50 value of 25 microM. DMHS increased internucleosomal DNA fragmentation in a time-dependent manner. The cell death by DMHS was partially prevented by the caspase inhibitor, zVAD-fmk. DMHS caused activation of caspases such as caspase-3, -8, and -9. Immunoblot experiments revealed that DMHS-induced apoptosis was associated with the induction of Bax expression. The release of cytochrome c from mitochondria into the cytosol was increased in response to DMHS. Taken together, our present results indicated that DMHS leads to apoptotic cell death in HL-60 cells through increased Bax expression and release of cytochrome c into cytosol and may be considered as a good candidate for a cancer chemopreventive agent in humans.