Protein serine/threonine phosphatase PPEF-1 suppresses genotoxic stress response via dephosphorylation of PDCD5.

Programmed cell death 5 (PDCD5) is believed to play a crucial role in p53 activation; however, the underlying mechanism of how PDCD5 function is regulated during apoptosis remains obscure. Here, we report that the serine/threonine phosphatase PPEF-1 interacts with and dephosphorylates PDCD5 at Ser-119, which leads to PDCD5 destabilization. Overexpression of wild-type PPEF-1, but not inactive PPEF-1D172N, efficiently suppressed CK2α-mediated stabilization of PDCD5 and p53-mediated apoptosis in response to etoposide (ET). Conversely, PPEF-1 knockdown further enhanced genotoxic stress responses. Notably, PPEF-1 suppressed p53-mediated genotoxic stress response via negative regulation of PDCD5. We also determined that overexpression of wild-type PPEF-1, but not inactive PPEF-1D172N, significantly increased tumorigenic growth and chemoresistance of A549 human lung carcinoma cells. Collectively, these data demonstrate that PPEF-1 plays a pivotal role in tumorigenesis of lung cancer cells by reducing PDCD5-mediated genotoxic stress responses.

Delphinidin induces apoptosis via cleaved HDAC3-mediated p53 acetylation and oligomerization in prostate cancer cells.

Delphinidin is a major anthocyanidin compound found in various fruits. It has anti-inflammatory, anti-oxidant, and various other biological activities. In this study, we identified the epigenetic modulators that mediate the apoptotic effect of delphinidin in human prostate cancer cells. We found that treatment of LNCaP cells (a p53 wild-type, human prostate cancer cell line) with delphinidin increased caspase-3, -7, and -8 activity, whereas it decreased histone deacetylase activity. Among class I HDACs, the activity of HDAC3 was specifically inhibited by delphinidin. Moreover, the induction of apoptosis by delphinidin was dependent on caspase-mediated cleavage of HDAC3, which results in the acetylation and stabilization of p53. We also observed that delphinidin potently upregulated pro-apoptotic genes that are positively regulated by p53, and downregulated various anti-apoptotic genes. Taken together, these results show that delphinidin induces p53-mediated apoptosis by suppressing HDAC activity and activating p53 acetylation in human prostate cancer LNCaP cells. Therefore, delphinidin may be useful in the prevention of prostate cancer.

Erratum to: Gecko proteins induce the apoptosis of bladder cancer 5637 cells by inhibiting Akt and activating the intrinsic caspase cascade.

The BMB Reports would like to correct in the reference of BMB Rep. 48(9), 531-536 titled "Gecko proteins induce the apoptosis of bladder cancer 5637 cells by inhibiting Akt and activating the intrinsic caspase cascade". The ACKNOWLEDGEMENTS should be corrected as follows, "This work was supported by the National Research Foundation of Korea (NRF-2010-0009086, NRF-2012R1A1A2039992, and 2012M3A9C7050184) and the Brain Busan 21 Project." and not "This work was partially supported by the National Research Foundation of Korea (NRF-2010-0009086, NRF-2003-003-C00110, and 2012M3A9C7050184) and the Brain Busan 21 Project." The online version reflects this change.

Gecko proteins induce the apoptosis of bladder cancer 5637 cells by inhibiting Akt and activating the intrinsic caspase cascade.

Gecko proteins have long been used as anti-tumor agents in oriental medicine, without any scientific background. Although anti-tumor effects of Gecko proteins on several cancers were recently reported, their effect on bladder cancer has not been investigated. Thus, we explored the anti-tumor effect of Gecko proteins and its cellular mechanisms in human bladder cancer 5637 cells. Gecko proteins significantly reduced the viability of 5637 cells without any cytotoxic effect on normal cells. These proteins increased the Annexin-V staining and the amount of condensed chromatin, demonstrating that the Gecko proteinsinduced cell death was caused by apoptosis. Gecko proteins suppressed Akt activation, and the overexpression of constitutively active form of myristoylated Akt prevented Gecko proteins-induced death of 5637 cells. Furthermore, Gecko proteins activated caspase 9 and caspase 3/7. Taken together, our data demonstrated that Gecko proteins suppressed the Akt pathway and activated the intrinsic caspase pathway, leading to the apoptosis of bladder cancer cells. [BMB Reports 2015; 48(9): 531-536].

DNAJB1 destabilizes PDCD5 to suppress p53-mediated apoptosis.

Although PDCD5 promotes p53-mediated apoptosis in various cancers, little is known about PDCD5 regulation. We recently found that DNAJB1 interacts with PDCD5 and induces the ubiquitin-dependent proteasomal degradation of PDCD5, thereby inhibiting p53-mediated apoptosis. To investigate these novel roles for PDCD5 and DNAJB1, we performed DNAJB1 mapping with PDCD5. PDCD5 specifically binds to the DNAJB1-D5 domain (Δ180-210), which was found to be essential for the stabilization of PDCD5. Further study showed that DNAJB1 post-translationally regulates PDCD5 stability. DNAJB1 ubiquitinated PDCD5 via a ubiquitin-mediated pathway. In human lung A549 cancer cells, DNAJB1 promoted the ubiquitination and degradation of PDCD5 and inhibited p53 activation. However, DNAJB1 knockdown in A549 cells increased the etoposide-induced activation of the p53-mediated apoptosis pathway and repressed cancer cell growth. Because this function was p53 dependent, DNAJB1 depletion increased the expression of p53-targeted apoptosis genes. In conclusion, we screened a novel PDCD5-associating protein, DNAJB1, by yeast two-hybrid screening and provided evidences that DNAJB1 targets PDCD5 to suppress p53-dependent apoptosis of cancer cells. Thus, we identified DNAJB1 as a negative regulator of PDCD5-mediated apoptosis and found that the apoptosis network of PDCD5 regulates cancer cell death.

Rubus coreanus Miquel inhibits acetylcholinesterase activity and prevents cognitive impairment in a mouse model of dementia.

To find acetylcholinesterase (AChE) inhibitors for the prevention of neurological disorders, such as Alzheimer's disease, ethanol extracts of promising traditional edible Korean plants were tested. Among them, Rubus coreanus Miquel extract exhibited the most significant AChE inhibitory activity. The effect of R. coreanus extract on trimethyltin-induced memory impairment in mice was investigated using Y-maze and passive avoidance tests. Our results showed that administration of R. coreanus extract significantly improved alternation behavior and step-through latency. In addition, R. coreanus extract was sequentially fractionated, and the purified constituent was determined to be 3,4,5-trihydroxybenzoic acid.

PKA negatively regulates PP2Cß to activate NF-κB-mediated inflammatory signaling.

Protein phosphatase 2Cß (PP2Cß) was found to act as a negative regulator of NF-κB-mediated inflammatory signaling; however, its regulatory mechanism has not been examined. Here, we show that protein kinase A (PKA) phosphorylates the PP2Cß, which was inhibited by PKA-specific inhibitor, H89. Mutation analysis of serine residues in PP2Cß revealed that Ser-195 in PP2Cß is phosphorylated by PKA. Importantly, PKA inhibition by H89 abrogated the Forskolin-induced destabilization of PP2Cß against ubiquitin-dependent proteosomal degradation pathway. Furthermore, H89 treatment efficiently reversed the negative effect of Forskolin on the anti-inflammatory function of PP2Cß. Collectively, these data suggest that PKA destabilizes PP2Cß upon inflammatory stimuli via phosphorylation of Ser-195 in PP2Cß.

Gecko Proteins Exert Anti-Tumor Effect against Cervical Cancer Cells Via PI3-Kinase/Akt Pathway.

Anti-tumor activity of the proteins from Gecko (GP) on cervical cancer cells, and its signaling mechanisms were assessed by viable cell counting, propidium iodide (PI) staining, and Western blot analysis. GP induced the cell death of HeLa cells in a dose-dependent manner while it did not affect the viability of normal cells. Western blot analysis showed that GP decreased the activation of Akt, and co-administration of GP and Akt inhibitors synergistically exerted anti-tumor activities on HeLa cells, suggesting the involvement of PI3-kinase/Akt pathway in GP-induced cell death of the cancer cells. Indeed, the cytotoxic effect of GP against HeLa cells was inhibited by overexpression of constituvely active form of Akt in HeLa cells. The candidates of the functional proteins in GP were analyzed by Mass-spectrum. Taken together, our results suggest that GP elicits anti-tumor activity against HeLa cells by inhibition of PI3-kinase/Akt pathway.

Gallic acid, a histone acetyltransferase inhibitor, suppresses ß-amyloid neurotoxicity by inhibiting microglial-mediated neuroinflammation.

SCOPE: We examined the biological effect of gallic acid (GA) as a nuclear factor (NF)-κB acetyltransferase inhibitor on microglial-mediated ß-amyloid neurotoxicity and restorative effects on the Aß-induced cognitive dysfunction. METHODS AND RESULTS: The protective effects of GA on the survival of neuronal cells were assessed with an MTT assay and a co-culture system. For the co-culture experiments, both BV-2 and primary microglia cells were treated with GA prior to Aß stimulation, and conditioned media were transferred to Neuro-2A cells. The mRNA and protein levels of inflammatory cytokines in both microglia and Neuro-2A cells were assessed with real-time polymerase chain reaction and western blotting. Inhibition of nuclear factor kappa B (NF-κB) acetylation with GA treatment resulted in reduced cytokine production in microglia cells and protection of neuronal cells from Aß-induced neurotoxicity. Furthermore, we observed a restorative effect of GA on Aß-induced cognitive dysfunction in mice with Y-maze and passive avoidance tests. Finally, we found that GA treatment efficiently blocked neuronal cell death by downregulating the expression of cytokines and the in vivo levels of NF-κB acetylation. CONCLUSION: These results suggest that selective inhibition of NF-κB acetylation by the histone acetyltransferase inhibitor GA is a possible therapeutic approach for alleviating the inflammatory progression of Alzheimer disease.

Neuroprotective effects of Eriobotrya japonica against ß-amyloid-induced oxidative stress and memory impairment.

The generation of oxygen free radicals and oxidative damage is believed to be involved in the pathogenesis of neurodegenerative disorders. Eriobotrya japonica has been used to treat several diseases in East Asia. In this study, we investigated the protective effect of an E. japonica extract against Aß peptide-induced oxidative stress. The 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay demonstrated that the E. japonica extract scavenged approximately 40% of DPPH radicals. Also, treatment of the E. japonica extract inhibited Aß(1-42)-mediated neuronal cell death. Furthermore, treatment of E. japonica extract efficiently suppressed the increase in intracellular ROS triggered by the Aß(1-42) peptide. Importantly, mice pre-treated with the E. japonica extract showed restoration of alternation behavior and reversal of Aß(1-42)-induced memory impairment. Consequently, the E. japonica extract substantially inhibited the increase in lipid peroxidation and restored superoxide dismutase activity. These results suggest that E. japonica protects from oxidative stress and cognitive deficits induced by the Aß peptide.

Histone deacetylase 3 is selectively involved in L3MBTL2-mediated transcriptional repression.

This is the first report that L(3)mbt-like 2 (L3MBTL2) specifically interacts with the histone deacetylase domain of histone deacetylase 3 (HDAC3) via its MBT domain. Here, we show that L3MBTL2 selectively interacts with HDAC3, but not other class I HDACs. An in vitro peptide-binding assay demonstrated the specific association of HDAC3 with methylated histone-K20 tail and L3MBTL2. Furthermore, depletion of HDAC3 resulted in a decrease of methylated K20-H4, as well as an increase in acetylated histone H3. Consequently, HDAC3 knock-down selectively suppressed L3MBTL2-mediated transcriptional repression. Taken together, our results reveal the concerted action of both HDAC3 and L3MBTL2 in histone deacetylation and methylation-dependent transcriptional repression.

Gallic acid suppresses lipopolysaccharide-induced nuclear factor-kappaB signaling by preventing RelA acetylation in A549 lung cancer cells.

Although multiple studies have revealed that gallic acid plays an important role in the inhibition of malignant transformation, cancer development, and inflammation, the molecular mechanism of gallic acid in inflammatory diseases is still unclear. In this study, we identified gallic acid from Rosa rugosa as a histone acetyltransferase (HAT) inhibitor with global specificity for the majority of HAT enzymes, but with no activity toward epigenetic enzymes including sirtuin (silent mating type information regulation 2 homologue) 1 (S. cerevisiae), histone deacetylase, and histone methyltransferase. Enzyme kinetic studies indicated that gallic acid uncompetitively inhibits p300/CBP-dependent HAT activities. We found that gallic acid inhibits p300-induced p65 acetylation, both in vitro and in vivo, increases the level of cytosolic IkappaBalpha, prevents lipopolysaccharide (LPS)-induced p65 translocation to the nucleus, and suppresses LPS-induced nuclear factor-kappaB activation in A549 lung cancer cells. We have also shown that gallic acid treatment inhibits the acetylation of p65 and the LPS-induced serum levels of interleukin-6 in vivo. Importantly, gallic acid generally inhibited inflammatory responses caused by other stimuli, including LPS, IFN-gamma, and interleukin-1beta, and further downregulated the expression of nuclear factor-kappaB-regulated antiapoptotic genes. These results show the crucial role of acetylation in the development of inflammatory diseases.

Epigallocatechin-3-gallate, a histone acetyltransferase inhibitor, inhibits EBV-induced B lymphocyte transformation via suppression of RelA acetylation.

Because the p300/CBP-mediated hyperacetylation of RelA (p65) is critical for nuclear factor-kappaB (NF-kappaB) activation, the attenuation of p65 acetylation is a potential molecular target for the prevention of chronic inflammation. During our ongoing screening study to identify natural compounds with histone acetyltransferase inhibitor (HATi) activity, we identified epigallocatechin-3-gallate (EGCG) as a novel HATi with global specificity for the majority of HAT enzymes but with no activity toward epigenetic enzymes including HDAC, SIRT1, and HMTase. At a dose of 100 micromol/L, EGCG abrogates p300-induced p65 acetylation in vitro and in vivo, increases the level of cytosolic IkappaBalpha, and suppresses tumor necrosis factor alpha (TNFalpha)-induced NF-kappaB activation. We also showed that EGCG prevents TNFalpha-induced p65 translocation to the nucleus, confirming that hyperacetylation is critical for NF-kappaB translocation as well as activity. Furthermore, EGCG treatment inhibited the acetylation of p65 and the expression of NF-kappaB target genes in response to diverse stimuli. Finally, EGCG reduced the binding of p300 to the promoter region of interleukin-6 gene with an increased recruitment of HDAC3, which highlights the importance of the balance between HATs and histone deacetylases in the NF-kappaB-mediated inflammatory signaling pathway. Importantly, EGCG at 50 micromol/L dose completely blocks EBV infection-induced cytokine expression and subsequently the EBV-induced B lymphocyte transformation. These results show the crucial role of acetylation in the development of inflammatory-related diseases.

Antiinflammatory effect of lactic acid bacteria: inhibition of cyclooxygenase-2 by suppressing nuclear factor-kappaB in Raw264.7 macrophage cells.

Lactobacillus casei 3260 (L. casei 3260) was evaluated in relation to the inflammatory response mediated by lipopolysaccharide (LPS)-induced nuclear factor-kappaB (NF-kappaB) and cyclooxygenase-2 (COX-2) expression in Raw264.7 macrophage cells. The treatment of Raw264.7 cells with L. casei 3260 significantly inhibited the secretion of tumor necrosis factor-alpha (TNF-alpha) and prostaglandins E2 (PGE2), followed by suppression of COX-2. To clarify the molecular mechanism, the inhibitory effect of L. casei 3260 on the NF-kappaB signaling pathway was examined based on the luciferase reporter activity. Although the treatment of Raw264.7 cells with L. casei 3260 did not affect the transcriptional activity of NF-kappaB, it did inhibit NF-kappaB activation, as determined by the cytosolic p65 release and degradation of I-kappaBalpha. Therefore, these findings suggest that the suppression of COX-2 through inhibiting the NF-kappaB activation by LPS may be associated with the antiinflammatory effects of L. casei 3260 on Raw264.7 cells.

The Role and Significance of Preoperative Breast MRI in the Setting of Breast Cancer / 한국유방암학회지

PURPOSE: Accurate preoperative assessment of breast cancer is important to determine the extent of disease and the plan for surgery. The purpose of this study was to evaluate the efficacy of preoperative breast magnetic resonance imaging (MRI) in breast cancer patients. METHODS: Between January 2001 and October 2007, 457 consecutive patients who had undergone surgical treatment for breast cancer were retrospectively studied. We compared 303 patients from the non-preoperative MRI group (group A) to 154 patients from the preoperative MRI group (group B). The impact of preoperative MRI was evaluated for each patient with regard to changes in therapeutic intervention. RESULTS: MRI alone revealed 17 new lesions. The results of the MRI led to a change in 9.1% of the planned surgical procedures. Tumor size was more accurately defined in patients undergoing MRI than in those undergoing ultrasound imaging. CONCLUSION: Breast MRI could be recommended as a preoperative diagnostic procedure in patients allocated to receive breast conservation surgery, because MRI may reveal unsuspected multifocal or multicentric tumors or carcinoma infiltrations and may result in changes in therapy.

Enzymatic synthesis and characterization of arbutin glucosides using glucansucrase from Leuconostoc mesenteroides B-1299CB.

Two arbutin glucosides were synthesized via the acceptor reaction of a glucansucrase from Leuconostoc mesenteroides B-1299CB with arbutin and sucrose. The glucosides were purified by Bio-gel P-2 column chromatography and high-performance liquid chromatography, and the structures were elucidated as 4-hydroxyphenyl beta-isomaltoside (arbutin-G1), 4-hydroxyphenyl beta-isomaltotrioside (arbutin-G2), according to the results of (1)H, (13)C, heteronuclear single-quantum coherence, (1)H-(1)H COSY, and heteronuclear multiple-bond correlation analyses. Arbutin glucoside (4-hydroxyphenyl beta-isomaltoside) exhibited slower effects on 1,1-diphenyl-2-picrylhydrazyl radical scavenging and similar effects on tyrosinase inhibition, and increased inhibitory effect on matrix metalloproteinase-1 production induced by UVB than arbutin.

Ameliorating effect of Gardenia jasminoides extract on amyloid beta peptide-induced neuronal cell deficit.

The brains of Alzheimer's disease (AD) patients are characterized by large deposits of amyloid beta peptide (Abeta). Abeta is known to increase free radical production in nerve cells, leading to cell death that is characterized by lipid peroxidation, free radical formation, protein oxi-dation, and DNA/RNA oxidation. In this study, we selected an extract of Gardenia jasminoides by screening, and investigated its ameliorating effects on Abeta-induced oxidative stress using PC12 cells. The effects of the extract were evaluated using the 2,7 -dichlorofluorescein diacetate (DCF-DA) assay and the 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay. To find the active component, the ethanol extract was partitioned with hexane, chloroform, and ethyl acetate, respectively, and the active component was purified by silica-gel column chromatography and HPLC. The results suggested that Gardenia jasminoides extract can reduce the cytotoxicity of Abeta in PC 12 cells, possibly by reducing oxidative stress.

Ferulic acid supplementation prevents trimethyltin-induced cognitive deficits in mice.

This study's objective was to clarify the ameliorative effects ferulic acid (4-hydroxy-3-methoxycinnamic acid) has against cognitive deficits and ChAT activation in trimethyltin (TMT) induced, memory injured mice following a 28-d ferulic acid treatment. After administering TMT for 3 d, each mouse performed Y-maze and passive avoidance tests to check immediate working memory performance and cognitive function. The results showed that ferulic acid administration attenuated TMT-induced memory injury and a decline in ChAT activity in the mice. This suggests that ferulic acid might be useful for preventing cognitive dysfunction as well as for boosting the activation of ChAT in dementia.

The Effect of alpha-Melanocyte Stimulating Hormone on Renal Ischemia Reperfusion Injury / 대한이식학회지

PURPOSE: In ischemia-reperfusion induced renal injuries, cytokines, chemoattractant chemokines, adhesion molecules and nitric oxide play an important role. alpha-Melanocyte stimulating hormone (alpha-MSH) is a potent anti-inflammatory cytokine so the therapeutic effect of alpha-MSH on an ischemia-reperfusion induced acute renal failure is to be evaluated. METHODS: 40 male Spraque-Dawley rats were prepared for the experiment, they were classified into three classes (Sham, ischemic control and alpha-MSH injection). Both renal pedicles were clamped for 45 minutes. alpha-MSH (50 microgram) was injected intravenously three times, immediately before ischemia and reperfusion and 18 hour after reperfusion. Serum creatinine and histologic changes were analyzed between groups (Sham (n=6), ischemic control group (n=15), and alpha-MSH group (n=19)). RESULTS: Serum creatinine level decreased significantly at 24 hours after reperfusion in alpha-MSH treated animals (SCr24 0.78+/-0.23 mg/dL, 4.21+/-1.14 mg/dL, 3.01+/-1.19 mg/dL, repectively (P=0.008)), especially serum creatinine level at 48 hours after reperfusion much more dicreased in alpha-MSH group (SCr48 0.67+/-0.16 mg/dL, 4.21+/-2.03 mg/dL, 1.15+/-1.11 mg/dL, repectively (P=0.004)). Tubular neccrosis and neutrophil infiltration decreased signigicantly in alpha-MSH treated group (P=0.001). Mortality was noted 33.3% only at ischemic conrol group. CONCLUSION: we demonstrate the fact that alpha-MSH has protective role on ischemic renal injury and improves survival rates.

Inhibitory effect of Aucubin isolated from Eucommia ulmoides against UVB-induced matrix metalloproteinase-1 production in human skin fibroblasts.

Of 30 herbal plants tested, the methanol extracts of Eucommia ulmoides (52%), Evodia officinalis (45%), and Pleuropterus multiflorus (41%) each showed a potent inhibitory effect on the matrix metalloproteinase-1 (MMP-1) production in ultraviolet B (UVB)-irradiated human fibroblasts. Aucubin was isolated as the MMP-1 inhibitor from E. ulmoides, and significantly suppressed the production of MMP-1 by nearly 57% compared to the control. It also reduced MMP-1 mRNA expression. These results suggest that aucubin is a photoprotective phytochemical, and could be used as a potential agent in preventing photoaging.