Wild simulated ginseng activates mouse macrophage, RAW264.7 cells through TRL2/4-dependent activation of MAPK, NF-κB and PI3K/AKT pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Ginseng (Panax ginseng Meyer) is a very well-known traditional herbal medicine that has long been used to enhance the body's immunity. Because it is a type of ginseng, it is believed that wild simulated ginseng (WSG) also has immune-enhancing activity. However, study on the immune-enhancing activity of WSG is quite insufficient compared to ginseng. AIM OF THE STUDY: In this study, we evaluated immune-enhancing activity of WSG through macrophage activation to provide a scientific basis for the immune enhancing activity of WSG. MATERIALS AND METHODS: The effect of WSG on viability of RAW264.7 cells was evaluated by MTT assay. The NO level was measured by Griess reagent. The expression levels of mRNA or protein in WSG-treated RAW264.7 cells were analyzed by RT-PCR and Western blot, respectively. RESULTS: WSG increased the production of immunomodulators such as NO, iNOS, COX-2, IL-1ß, IL-6 and TNF-α and activated phagocytosis in mouse macrophages RAW264.7 cells. Inhibition of TLR2 and TLR4 reduced the production of immunomodulators induced by WSG. WSG activated MAPK, NF-κB and PI3K/AKT signaling pathways, and inhibition of such signaling activation blocked WSG-mediated production of immunomodulators. In addition, activation of MAPK, NF-κB and PI3K/AKT signaling pathways by WSG was reversed by TLR2 or TLR4 inhibition. CONCLUSION: Based on the results of this study, WSG is thought to activate macrophages through the production of immunomodulators and phagocytosis activation through TLR2/4-dependent MAPK, NF-κB and PI3K/AKT signaling pathways. Therefore, it is thought that WSG have the potential to be used as an agent for enhancing immunity.

Whole-genome, transcriptome, and methylome analyses provide insights into the evolution of platycoside biosynthesis in Platycodon grandiflorus, a medicinal plant.

Triterpenoid saponins (TSs) are common plant defense phytochemicals with potential pharmaceutical properties. Platycodon grandiflorus (Campanulaceae) has been traditionally used to treat bronchitis and asthma in East Asia. The oleanane-type TSs, platycosides, are a major component of the P. grandiflorus root extract. Recent studies show that platycosides exhibit anti-inflammatory, antiobesity, anticancer, antiviral, and antiallergy properties. However, the evolutionary history of platycoside biosynthesis genes remains unknown. In this study, we sequenced the genome of P. grandiflorus and investigated the genes involved in platycoside biosynthesis. The draft genome of P. grandiflorus is 680.1 Mb long and contains 40,017 protein-coding genes. Genomic analysis revealed that the CYP716 family genes play a major role in platycoside oxidation. The CYP716 gene family of P. grandiflorus was much larger than that of other Asterid species. Orthologous gene annotation also revealed the expansion of ß-amyrin synthases (bASs) in P. grandiflorus, which was confirmed by tissue-specific gene expression. In these expanded gene families, we identified key genes showing preferential expression in roots and association with platycoside biosynthesis. In addition, whole-genome bisulfite sequencing showed that CYP716 and bAS genes are hypomethylated in P. grandiflorus, suggesting that epigenetic modification of these two gene families affects platycoside biosynthesis. Thus whole-genome, transcriptome, and methylome data of P. grandiflorus provide novel insights into the regulation of platycoside biosynthesis by CYP716 and bAS gene families.

Wood-cultivated ginseng exerts anti-inflammatory effect in LPS-stimulated RAW264.7 cells.

Ginseng (Panax ginseng) has been reported to exert an anti-inflammatory activity in a variety of inflammatory condition. However, inflammation-regulatory activity of wood-cultivated ginseng has not been thoroughly evaluated. In this study, we evaluated the anti-inflammatory effect of wood-cultivated ginseng (WCG) and elucidated the potential mechanisms in LPS-stimulated RAW264.7 cells. WCG-O dose-dependently suppressed NO and PGE2 production in LPS-stimulated RAW264.7 cells. In addition, WCG-O attenuated LPS-mediated overexpression of iNOS and COX-2. In addition, WCG-O blocked the expression of TNF-α and IL-1ß. WCG-O inhibited the activation of IκK-α/ß, the phosphorylation of IκB-α, and degradation of IκB-α, which results in the inhibition of p65 nuclear accumulation and NF-κB activation. In addition, WCG-O suppressed the activation of ERK1/2, p38 and JNK, which results in the inhibition of ATF2 nuclear accumulation. These results indicate that WCG-O may exert anti-inflammatory activity by inhibiting NF-κB and MAPK signaling. From these findings, WCG-O has potential to be a candidate for the development of chemopreventive or therapeutic agents for the inflammatory diseases.

Cytotoxic activity of the twigs of Cinnamomum cassia through the suppression of cell proliferation and the induction of apoptosis in human colorectal cancer cells.

BACKGROUND: Because twigs of Cinnamomum cassia (TC) have been reported to exert anti-cancer activity, the mechanistic study for TC's anti-cancer activity is required. Thus, we elucidated the potential molecular mechanism of TC's anti-proliferative effect and the induction of apoptosis in human colorectal cancer cells. METHODS: How water extracts form TC (TC-HW) was used in this study. Anti-cell proliferative effect of TC-HW was evaluated by MTT assay. The change of protein or mRNA level by TC-HW was evaluated by Western blot and RT-RCR, respectively. The promoter construct for ATF3, NF-κB, TOP-FLASH or FOP-FLASH was used for the investigation of the transcriptional activity for ATF3, NF-κB or Wnt. siRNA for ATF3 or p65 was used for the knockdown of ATF3 and p65. RESULTS: TC-HW reduced the cell viability in human colorectal cancer cells. TC-HW decreased cyclin D1 protein level through cyclin D1 degradation via GSK3ß-dependent threonine-286 (T286) phosphorylation of cyclin D1, indicating that cyclin D1 degradation may contribute to TC-HW-mediated decrease of cyclin D1 protein level. TC-HW downregulated the expression of cyclin D1 mRNA level and inhibited Wnt activation through the downregulation of ß-catenin and TCF4 expression, indicating that inhibition of cyclin D1 transcription may also result in TC-HW-mediated decrease of cyclin D1 protein level. In addition, TC-HW was observed to induce apoptosis through ROS-dependent DNA damage. TC-HW-induced ROS increased NF-κB and ATF3 activation, and inhibition of NF-κB and ATF3 activation attenuated TC-HW-mediated apoptosis. CONCLUSIONS: Our results suggest that TC-HW may suppress cell proliferation through the downregulation of cyclin D1 via proteasomal degradation and transcriptional inhibition, and may induce apoptosis through ROS-dependent NF-κB and ATF3 activation. These effects of TC-HW may contribute to the reduction of cell viability in human colorectal cancer cells. From these findings, TC-HW has potential to be a candidate for the development of chemoprevention or therapeutic agents for human colorectal cancer.

Vitex rotundifolia Fruit Suppresses the Proliferation of Human Colorectal Cancer Cells through Down-regulation of Cyclin D1 and CDK4 via Proteasomal-Dependent Degradation and Transcriptional Inhibition.

Viticis Fructus (VF) as the dried fruit from Vitex rotundifolia L. used as a traditional medicine for treating inflammation, headache, migraine, chronic bronchitis, eye pain, and gastrointestinal infections has been reported to have antiproliferative effects against various cancer cells, including breast, lung and colorectal cancer cells. However, the molecular mechanisms by which VF mediates the inhibitory effect of the proliferation of cancer cells have not been elucidated in detail. In this study, we investigated the molecular mechanism of VF on the down-regulation of cyclin D1 and CDK4 level associated with cancer cell proliferation. VF suppressed the proliferation of human colorectal cancer cell lines such as HCT116 and SW480. VF induced decrease in cyclin D1 and CDK4 in both protein and mRNA levels. However, the protein levels of cyclin D1 and CDK4 were decreased by VF at an earlier time than the change of mRNA levels; rather it suppressed the expression of cyclin D1 and CDK4 via the proteasomal degradation. In cyclin D1 and CDK4 degradation, we found that Thr286 phosphorylation of cyclin D1 plays a pivotal role in VF-mediated cyclin D1 degradation. Subsequent experiments with several kinase inhibitors suggest that VF-mediated degradation of cyclin D1 may be dependent on GSK3[Formula: see text] and VF-mediated degradation of CDK4 is dependent on ERK1/2, p38 and GSK3[Formula: see text]. In the transcriptional regulation of cyclin D1 and CDK4, we found that VF inhibited Wnt activation associated with cyclin D1 transcriptional regulation through TCF4 down-regulation. In addition, VF treatment down-regulated c-myc expression associated CDK4 transcriptional regulation. Our results suggest that VF has potential to be a candidate for the development of chemoprevention or therapeutic agents for human colorectal cancer.

Anticancer activity of calyx of Diospyros kaki Thunb. through downregulation of cyclin D1 via inducing proteasomal degradation and transcriptional inhibition in human colorectal cancer cells.

BACKGROUND: Although it has been reported to contain high polyphenols, the pharmacological studies of the calyx of Diospyros kaki Thunb (DKC) have not been elucidated in detail. In this study, we elucidated anti-cancer activity and potential molecular mechanism of DKC against human colorectal cancer cells. METHODS: Anti-cell proliferative effect of 70% ethanol extracts from the calyx of Diospyros kaki (DKC-E70) was evaluated by MTT assay. The effect of DKC-E70 on the expression of cyclin D1 in the protein and mRNA level was evaluated by Western blot and RT-PCR, respectively. RESULTS: DKC-E70 suppressed the proliferation of human colorectal cancer cell lines such as HCT116, SW480, LoVo and HT-29. Although DKC-E70 decreased cyclin D1 expression in protein and mRNA level, decreased level of cyclin D1 protein by DKC-E70 occurred at the earlier time than that of cyclin D1 mRNA, which indicates that DKC-E70-mediated downregulation of cyclin D1 protein may be a consequence of the induction of degradation and transcriptional inhibition of cyclin D1. In cyclin D1 degradation, we found that cyclin D1 downregulation by DKC-E70 was attenuated in presence of MG132. In addition, DKC-E70 phosphorylated threonine-286 (T286) of cyclin D1 and T286A abolished cyclin D1 downregulation by DKC-E70. We also observed that DKC-E70-mediated T286 phosphorylation and subsequent cyclin D1 degradation was blocked in presence of the inhibitors of ERK1/2, p38 or GSK3ß. In cyclin D1 transcriptional inhibition, DKC-E70 inhibited the expression of ß-catenin and TCF4, and ß-catenin/TCF-dependent luciferase activity. CONCLUSIONS: Our results suggest that DKC-E70 may downregulate cyclin D1 as one of the potential anti-cancer targets through cyclin D1 degradation by T286 phosphorylation dependent on ERK1/2, p38 or GSK3ß, and cyclin D1 transcriptional inhibition through Wnt signaling. From these findings, DKC-E70 has potential to be a candidate for the development of chemoprevention or therapeutic agents for human colorectal cancer.

A combination of biochemical and proteomic analyses reveals Bx-LEC-1 as an antigenic target for the monoclonal antibody 3-2A7-2H5-D9-F10 specific to the pine wood nematode.

Pine wilt disease (PWD) is one of the most devastating forest diseases in Asia and Europe. The pine wood nematode, Bursaphelenchus xylophilus, has been identified as the pathogen underlying PWD, although the pathology is not completely understood. At present, diagnosis and confirmation of PWD are time consuming tasks that require nematode extraction and microscopic examination. To develop a more efficient detection method for B. xylophilus, we first generated monoclonal antibodies (MAbs) specific to B. xylophilus. Among 2304 hybridoma fusions screened, a hybridoma clone named 3-2A7-2H5 recognized a single protein from B. xylophilus specifically, but not those from other closely related nematodes. We finally selected the MAb clone 3-2A7-2H5-D9-F10 (D9-F10) for further studies. To identify the antigenic target of MAb-D9-F10, we analyzed proteins in spots, fractions, or bands isolated from SDS-PAGE, two-dimensional electrophoresis, anion exchange chromatography, and immunoprecipitation via nano liquid chromatography electrospray ionization quadrupole ion trap mass spectrometry (nano-LC-ESI-Q-IT-MS). Peptides of galactose-binding lectin-1 of B. xylophilus (Bx-LEC-1) were commonly detected in several proteomic analyses, demonstrating that this LEC-1 is the antigenic target of MAb-D9-F10. The localization of MAb-D9-F10 immunoreactivities at the area of the median bulb and esophageal glands suggested that the Bx-LEC-1 may be involved in food perception and digestion. The Bx-LEC-1 has two nonidentical galactose-binding lectin domains important for carbohydrate binding. The affinity of the Bx-LEC-1 to D-(+)-raffinose and N-acetyllactosamine were much higher than that to L-(+)-rhamnose. Based on this combination of evidences, MAb-D9-F10 is the first identified molecular biomarker specific to the Bx-LEC-1.