INHAT subunit SET/TAF-Iß regulates PRC1-independent H2AK119 mono-ubiquitination via E3 ligase MIB1 in colon cancer.

SET/TAF-Iß, a subunit of the inhibitor of acetyltransferases (INHAT) complex, exhibits transcriptional repression activity by inhibiting histone acetylation. We find that SET/TAF-Iß regulates mono-ubiquitination of histone H2A at lysine 119 (H2AK119ub), which is involved in polycomb-mediated transcriptional repression, in HCT116 cells. In this report, we demonstrate that SET/TAF-Iß acts as an E2 ubiquitin-conjugating enzyme for PRC1-independent H2AK119ub. Furthermore, we identify that MIB1 is the E3 ligase partner for SET/TAF-Iß using LC-MS/MS and in vitro ubiquitination assays. Transcriptome analysis reveals that SET/TAF-Iß and MIB1 regulate the expression of genes related to DNA replication and cell cycle progression in HCT116 cells, and knockdown of either protein reduces proliferation of HCT116 cells by impeding cell cycle progression. Together, our study reveals a novel PRC1-independent epigenetic regulatory mechanism for H2AK119ub by SET/TAF-Iß and MIB1 in colon cancer.

FOXO1 degradation via G9a-mediated methylation promotes cell proliferation in colon cancer.

Posttranslational modifications of the Forkhead family transcription factor, FOXO1, have been known to have important regulatory implications in its diverse activities. Several types of modifications of FOXO1, including acetylation, phosphorylation, and ubiquitination, have been reported. However, lysine methylation of FOXO1 has not yet been identified. Here, we reported that FOXO1 is methylated by G9a at K273 residue in vitro and in vivo. Methylation of FOXO1 by G9a increased interaction between FOXO1 and a specific E3 ligase, SKP2, and decreased FOXO1 protein stability. In addition, G9a expression was increased by insulin and resulted in insulin-mediated FOXO1 degradation by K273 methylation. Tissue array analysis indicated that G9a was overexpressed and FOXO1 levels decreased in human colon cancer. Cell proliferation assays revealed that G9a-mediated FOXO1 methylation increased colon cancer cell proliferation. Fluorescence-activated cell sorting (FACS) analysis indicated that apoptosis rates were higher in the presence of FOXO1 than in FOXO1 knock-out cells. Furthermore, we found that G9a protein levels were elevated and FOXO1 protein levels were decreased in human colon cancer patients tissue samples. Here, we report that G9a specific inhibitor, BIX-01294, can regulate cell proliferation and apoptosis by inhibiting G9a-mediated FOXO1 methylation.

Methylation of Aurora kinase A by MMSET reduces p53 stability and regulates cell proliferation and apoptosis.

The histone methyltransferase multiple myeloma SET domain protein (MMSET/WHSC1) is highly expressed in diverse tumor types, and its expression appears to be involved in cell proliferation. In this study, we report that MMSET interacts with and methylates Aurora kinase A (AURKA). We show that MMSET-mediated methylation of AURKA induces interaction with p53 as well as enhanced kinase activity of AURKA, which results in the proteasomal degradation of p53. MMSET-mediated p53 degradation increases cell proliferation and results in oncogenic activity. Furthermore, knockdown of MMSET potently inhibits tumorigenic cells and renders them sensitive to growth inhibition by the therapeutic drug, alisertib (AURKA inhibitor). Taken together, our results suggest that MMSET is a regulator of p53 stability via methylation of AURKA in proliferating cells and might be a potential therapeutic target in solid tumors.

AURKA Suppresses Leukemic THP-1 Cell Differentiation through Inhibition of the KDM6B Pathway.

Aberrations in histone modifications are being studied in mixed-lineage leukemia (MLL)-AF9-driven acute myeloid leukemia (AML). In this study, we focused on the regulation of the differentiation of the MLL-AF9 type AML cell line THP-1. We observed that, upon phorbol 12-myristate 13-acetate (PMA) treatment, THP-1 cells differentiated into monocytes by down-regulating Aurora kinase A (AURKA), resulting in a reduction in H3S10 phosphorylation. We revealed that the AURKA inhibitor alisertib accelerates the expression of the H3K27 demethylase KDM6B, thereby dissociating AURKA and YY1 from the KDM6B promoter region. Using Flow cytometry, we found that alisertib induces THP-1 differentiation into monocytes. Furthermore, we found that treatment with the KDM6B inhibitor GSK-J4 perturbed the PMA-mediated differentiation of THP-1 cells. Thus, we discovered the mechanism of AURKA-KDM6B signaling that controls the differentiation of THP-1 cells, which has implications for biotherapy for leukemia.

An experimental study on providing a scientific evidence for seven-time alcohol-steaming of Rhei Rhizoma when clinically used.

BACKGROUND: Rhei Rhizoma (RR) has been widely used as laxative and processed to alter its therapeutic actions or reduce its side effects. In this study, we evaluated experimentally the clinical application guideline that RR should be alcohol-steamed seven times before being used in elderly patients, as described in Dongeuibogam, the most famous book on Korean traditional medicine. METHODS: Unprocessed RR (RR-U) was soaked in rice wine, steamed and then fully dried (RR-P1). The process was repeated four (RR-P4) or seven times (RR-P7). Reversed-phase high-performance liquid chromatography was used to determine the RR-U, RR-P1, RR-P4 and RR-P7 (RRs) constituents. To evaluate the effect of RRs on liver toxicity, human hepatoma cells (HepG2) were treated with RRs at 100 µg/mL for 4 h and then cell viabilities were measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. To confirm the effects in vivo, 5-week-old male Sprague-Dawley rats were treated with RRs at 3 g/kg/day for 21 days. Body weight and serum biochemical parameters were measured and liver histology was assessed. RESULTS: The levels of sennosides decreased in processed RRs in an iteration-dependent manner, while the emodin level was unaffected. In HepG2 cells, cell viability was reduced with RR-U, while the toxicity decreased according to the number of processing cycles. The changes in body weight, relative liver weight and liver enzymes of RR-U-treated rats were reduced in processed RRs-treated rats. Histopathological analysis indicated swelling and cholestasis improved following seven times alcohol-steaming cycles. CONCLUSIONS: These results provide experimental evidence that RR-P7 almost completely reduces RR hepatotoxicity.

Ginger improves cognitive function via NGF-induced ERK/CREB activation in the hippocampus of the mouse.

Ginger (the rhizome of Zingiber officinale Roscoe) has been used worldwide for many centuries in cooking and for treatment of several diseases. The main pharmacological properties of ginger include anti-inflammatory, antihyperglycemic, antiarthritic, antiemetic and neuroprotective actions. Recent studies demonstrated that ginger significantly enhances cognitive function in various cognitive disorders as well as in healthy brain. However, the biochemical mechanisms underlying the ginger-mediated enhancement of cognition have not yet been studied in normal or diseased brain. In the present study, we assessed the memory-enhancing effects of dried ginger extract (GE) in a model of scopolamine-induced memory deficits and in normal animals by performing a novel object recognition test. We found that GE administration significantly improved the ability of mice to recognize novel objects, indicating improvements in learning and memory. Furthermore, to elucidate the mechanisms of GE-mediated cognitive enhancement, we focused on nerve growth factor (NGF)-induced signaling pathways. NGF enzyme-linked immunosorbent assay analysis revealed that GE administration led to elevated NGF levels in both the mouse hippocampus and rat glioma C6 cells. GE administration also resulted in phosphorylation of extracellular-signal-regulated kinase (ERK) and cyclic AMP response element-binding protein (CREB), as revealed by Western blotting analysis. Neutralization of NGF with a specific NGF antibody inhibited GE-triggered activation of ERK and CREB in the hippocampus. Also, GE treatment significantly increased pre- and postsynaptic markers, synaptophysin and PSD-95, which are related to synapse formation in the brain. These data suggest that GE has a synaptogenic effect via NGF-induced ERK/CREB activation, resulting in memory enhancement.

6-Shogaol, an active constituent of ginger, attenuates neuroinflammation and cognitive deficits in animal models of dementia.

Recently, increased attention has been directed towards medicinal extracts as potential new drug candidates for dementia. Ginger has long been used as an important ingredient in cooking and traditional herbal medicine. In particular, ginger has been known to have disease-modifying effects in Alzheimer's disease (AD). However, there is no evidence of which constituents of ginger exhibit therapeutic effects against AD. A growing number of experimental studies suggest that 6-shogaol, a bioactive component of ginger, may play an important role as a memory-enhancing and anti-oxidant agent against neurological diseases. 6-Shogaol has also recently been shown to have anti-neuroinflammatory effects in lipopolysaccharide (LPS)-treated astrocytes and animal models of Parkinson's disease, LPS-induced inflammation and transient global ischemia. However, it is still unknown whether 6-shogaol has anti-inflammatory effects against oligomeric forms of the Aß (AßO) in animal brains. Furthermore, the effects of 6-shogaol against memory impairment in dementia models are also yet to be investigated. In this study, we found that administration of 6-shogaol significantly reduced microgliosis and astrogliosis in intrahippocampal AßO-injected mice, ameliorated AßO and scopolamine-induced memory impairment, and elevated NGF levels and pre- and post-synaptic marker in the hippocampus. All these results suggest that 6-shogaol may play a role in inhibiting glial cell activation and reducing memory impairment in animal models of dementia.

Sanguisorbae radix protects against 6-hydroxydopamine-induced neurotoxicity by regulating NADPH oxidase and NF-E2-related factor-2/heme oxygenase-1 expressions.

6-Hydroxydopamine (6-OHDA) produces neuronal cell damage by generating reactive oxygen species (ROS). The major mechanisms of protection against ROS-induced stress are inhibiting expression of ROS generating genes such as NADPH oxidase (NOX) and increasing expression of endogenous antioxidant genes such as heme oxygenase-1 (HO-1). This study investigated whether a standardized Sanguisorbae Radix extract (SRE), a medical herb commonly used in Asian traditional medicine, has a protective effect on 6-OHDA-induced cell toxicity by regulating ROS in SH-SY5Y cells. SRE at 10 and 50 µg/mL significantly reduced 6-OHDA-induced cell damage dose dependently in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and by Hoechst 33342 staining. SRE increased the B-cell lymphoma 2 (Bcl-2)/Bcl-2-associated X ratio and decreased cytochrome C release and caspase-3 activity. SRE also abolished 6-OHDA-induced ROS by inhibiting NOX expression and by inducing HO-1 expression via NF-E2-related factor-2 activation. Taken together, these results demonstrate that SRE has protective effects against 6-OHDA-induced cell death by regulating ROS in SH-SY5Y cells.

Protective effect of Cyperi rhizoma against 6-hydroxydopamine-induced neuronal damage.

Cyperi rhizoma, the rhizome of Cyperus rotundus L. (Family Cyperaceae), is a well-known functional food and traditional herbal medicine in Korea. It has been reported that Cyperi rhizoma has antioxidant and free radical scavenging activities that play a major role in protection of neurodegenerative disorders, such as Parkinson's disease (PD). In the present study, the neuroprotective effects of a water extract of Cyperi rhizoma (CRE) against 6-hydroxydopamine (6-OHDA)-induced neuronal damage were evaluated in an experimental model of PD. In PC12 cells, CRE showed a significant protective effect on cell viability at 50 and 100 microg/mL. CRE inhibited generation of reactive oxygen species and nitric oxide, reduction of mitochondrial membrane potential, and caspase-3 activity, which were induced by 6-OHDA. CRE also showed a significant protective effect against damage to dopaminergic neurons in primary mesencephalic culture. These results suggest that CRE has neuroprotective effects against 6-OHDA-induced toxicity through antioxidant and anti-apoptotic activities in an in vitro PD model.