Storax Attenuates Cardiac Fibrosis following Acute Myocardial Infarction in Rats via Suppression of AT1R-Ankrd1-P53 Signaling Pathway.

Myocardial fibrosis following acute myocardial infarction (AMI) seriously affects the prognosis and survival rate of patients. This study explores the role and regulation mechanism of storax, a commonly used traditional Chinese medicine for treatment of cardiovascular diseases, on myocardial fibrosis and cardiac function. The AMI rat model was established by subcutaneous injection of Isoproterenol hydrochloride (ISO). Storax (0.1, 0.2, 0.4 g/kg) was administered by gavage once/d for 7 days. Electrocardiogram, echocardiography, hemodynamic and cardiac enzyme in AMI rats were measured. HE, Masson, immunofluorescence and TUNEL staining were used to observe the degree of pathological damage, fibrosis and cardiomyocyte apoptosis in myocardial tissue, respectively. Expression of AT1R, CARP and their downstream related apoptotic proteins were detected by WB. The results demonstrated that storax could significantly improve cardiac electrophysiology and function, decrease serum cardiac enzyme activity, reduce type I and III collagen contents to improve fibrosis and alleviate myocardial pathological damage and cardiomyocyte apoptosis. It also found that storax can significantly down-regulate expression of AT1R, Ankrd1, P53, P-p53 (ser 15), Bax and cleaved Caspase-3 and up-regulate expression of Mdm2 and Bcl-2. Taken together, these findings indicated that storax effectively protected cardiomyocytes against myocardial fibrosis and cardiac dysfunction by inhibiting the AT1R-Ankrd1-P53 signaling pathway.

Sestrin2 as a Potential Target for Regulating Metabolic-Related Diseases.

Sestrin2 is a highly conserved protein that can be induced under a variety of stress conditions, including DNA damage, oxidative stress, endoplasmic reticulum (ER) stress, and metabolic stress. Numerous studies have shown that the AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) signaling pathway has a crucial role in the regulation of metabolism. Sestrin2 regulates metabolism via a number of pathways, including activation of AMPK, inhibition of the mTOR complex 1 (mTORC1), activation of mTOR complex 2 (mTORC2), inhibition of ER stress, and promotion of autophagy. Therefore, modulation of Sestrin2 activity may provide a potential therapeutic target for the prevention of metabolic diseases such as insulin resistance, diabetes, obesity, non-alcoholic fatty liver disease, and myocardial ischemia/reperfusion injury. In this review, we examined the regulatory relationship between Sestrin2 and the AMPK/mTOR signaling pathway and the effects of Sestrin2 on energy metabolism.

Molecular features of exceptional response to neoadjuvant anti-androgen therapy in high-risk localized prostate cancer.

High-risk localized prostate cancer (HRLPC) is associated with a substantial risk of recurrence and disease mortality. Recent clinical trials have shown that intensifying anti-androgen therapies administered before prostatectomy can induce pathologic complete responses or minimal residual disease, called exceptional response, although the molecular determinants of these clinical outcomes are largely unknown. Here, we perform whole-exome and transcriptome sequencing on pre-treatment multi-regional tumor biopsies from exceptional responders (ERs) and non-responders (NRs, pathologic T3 or lymph node-positive disease) to intensive neoadjuvant anti-androgen therapies. Clonal SPOP mutation and SPOPL copy-number loss are exclusively observed in ERs, while clonal TP53 mutation and PTEN copy-number loss are exclusively observed in NRs. Transcriptional programs involving androgen signaling and TGF-ß signaling are enriched in ERs and NRs, respectively. These findings may guide prospective validation studies of these molecular features in large HRLPC clinical cohorts treated with neoadjuvant anti-androgens to improve patient stratification.

Stem-loop binding protein and metal carcinogenesis.

Pre-mRNA processing of the replication-dependent canonical histone mRNAs requires an endonucleolytic cleavage immediately after a conserved stem loop structure which occurs before RNA Pol II encounters any poly(A) signal. Thus, in contrast to all other eukaryotic mRNAs, the canonical histone mRNAs are not polyadenylated in their 3' ends. The binding of stem-loop binding protein (SLBP) to the stem loop structure of the histone mRNAs is required for this process. SLBP is also involved in regulation of histone mRNA nuclear export, degradation, and translation. Depletion of SLBP has been shown to induce polyadenylation of histone mRNAs and alteration of histone protein levels, which are considered to contribute to the observed aberrant cell cycle progress and genomic instability resulting from the loss of SLBP function. Recent studies have demonstrated that some heavy metal carcinogens, including arsenic and nickel, can induce the loss of SLBP and the gain of polyadenylation of canonical histone mRNAs. Polyadenylated canonical histone H3 can result in abnormal transcription, cell cycle arrest, genomic instability, and cell transformation, which links SLBP depletion and subsequent histone mRNA misprocessing to cancer. This review seeks to briefly summarize what is known about regulation of SLBP expression, consequences of SLBP depletion, its roles in cancer-related end points, with particular focus on metal-induced SLBP depletion and the potential of SLBP depletion as a new mechanism for metal-induced carcinogenesis.

Kaempferol alleviates human endothelial cell injury through circNOL12/miR-6873-3p/FRS2 axis.

BACKGROUND: Atherosclerosis, inflammatory disease, is a major reason for cardiovascular diseases and stroke. Kaempferol (Kae) has been well-documented to have pharmacological activities in the previous studies. However, the detailed mechanisms by which Kae regulates inflammation, oxidative stress, and apoptosis in Human Umbilical Vein Endothelial Cells (HUVECs) remain unknown. METHODS AND RESULTS: The real-time quantitative polymerase chain reaction (RT-qPCR) was used to measure expression levels of circNOL12, nucleolar protein 12 (NOL12), miR-6873-3p, and Fibroblast growth factor receptor substrate 2 (FRS2) in HUVECs treated with either oxidized low-density lipoprotein (ox-LDL) alone or in combination with Kae. The cells viability was assessed by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-2H-tetrazol-3-ium bromide (MTT) assay. The inflammation and oxidative stress were assessed by checking inflammatory factors, Reactive Oxygen Species (ROS), Superoxide Dismutase (SOD), and Malondialdehyde (MDA) levels in ox-LDL-induced HUVECs. The apoptotic cells were quantified by flow cytometry assay. The western blot assay was used for measuring protein expression. The interaction relationship between miR-6873-3p and circNOL12 or FRS2 was analyzed by dual-luciferase reporter and RNA pull-down assays. Treatment with Kae could inhibit ox-LDL-induced the upregulation of circNOL12 in HUVECs. Importantly, Kae weakened ox-LDL-induced inflammation, oxidative stress, and apoptosis in HUVECs, which was abolished by overexpression of circNOL12. What's more, miR-6873-3p was a target of circNOL12 in HUVECs, and the upregulation of miR-6873-3p overturned circNOL12 overexpression-induced effects on HUVECs treated with ox-LDL and Kae. FRS2 was negatively regulated by miR-6873-3p in HUVECs. CONCLUSION: Kae alleviated ox-LDL-induced inflammation, oxidative stress, and apoptosis in HUVECs by regulating circNOL12/miR-6873-3p/FRS2 axis.

Changes in the Expression of TBP-2 in Response to Histone Deacetylase Inhibitor Treatment in Human Endometrial Cells.

Histone deacetylase inhibitors (HDACi) induce apoptosis preferentially in cancer cells by caspase pathway activation and reactive oxygen species (ROS) accumulation. Suberoylanilide hydroxamic acid (SAHA), a HDACi, increases apoptosis via altering intracellular oxidative stress through thioredoxin (TRX) and TRX binding protein-2 (TBP-2). Because ROS accumulation, as well as the redox status determined by TBP-2 and TRX, are suggested as possible mechanisms for endometriosis, we queried whether SAHA induces apoptosis of human endometrial cells via the TRX-TBP-2 system in endometriosis. Eutopic endometrium from participants without endometriosis, and ectopic endometrium from patients with endometriosis, was obtained surgically. Human endometrial stromal cells (HESCs) and Ishikawa cells were treated with SAHA and cell proliferation was assessed using the CCK-8 assay. Real-time PCR and Western blotting were used to quantify TRX and TBP-2 mRNA and protein expression. After inducing oxidative stress, SAHA was applied. Short-interfering TRX (SiTRX) transfection was performed to see the changes after TRX inhibition. The mRNA and protein expression of TBP-2 was increased with SAHA concentrations in HESCs significantly. The mRNA TBP-2 expression was decreased after oxidative stress, upregulated by adding 2.5 µM of SAHA. The TRX/TBP-2 ratio decreased, apoptosis increased significantly, and SiTRX transfection decreased with SAHA. In conclusion, SAHA induces apoptosis by modulating the TRX/TBP-2 system, suggesting its potential as a therapeutic agent for endometriosis.

BETs inhibition attenuates oxidative stress and preserves muscle integrity in Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) affects 1 in 3500 live male births. To date, there is no effective cure for DMD, and the identification of novel molecular targets involved in disease progression is important to design more effective treatments and therapies to alleviate DMD symptoms. Here, we show that protein levels of the Bromodomain and extra-terminal domain (BET) protein BRD4 are significantly increased in the muscle of the mouse model of DMD, the mdx mouse, and that pharmacological inhibition of the BET proteins has a beneficial outcome, tempering oxidative stress and muscle damage. Alterations in reactive oxygen species (ROS) metabolism are an early event in DMD onset and they are tightly linked to inflammation, fibrosis, and necrosis in skeletal muscle. By restoring ROS metabolism, BET inhibition ameliorates these hallmarks of the dystrophic muscle, translating to a beneficial effect on muscle function. BRD4 direct association to chromatin regulatory regions of the NADPH oxidase subunits increases in the mdx muscle and JQ1 administration reduces BRD4 and BRD2 recruitment at these regions. JQ1 treatment reduces NADPH subunit transcript levels in mdx muscles, isolated myofibers and DMD immortalized myoblasts. Our data highlight novel functions of the BET proteins in dystrophic skeletal muscle and suggest that BET inhibitors may ameliorate the pathophysiology of DMD.

Melatonin alters neuronal architecture and increases cysteine-rich protein 1 signaling in the male mouse hippocampus.

Neuronal plasticity describes changes in structure, function, and connections of neurons. The hippocampus, in particular, has been shown to exhibit considerable plasticity regarding both physiological and morphological functions. Melatonin, a hormone released by the pineal gland, promotes cell survival and dendrite maturation of neurons in the newborn brain and protects against neurological disorders. In this study, we investigated the effect of exogenous melatonin on neuronal architecture and its possible mechanism in the hippocampus of adult male C57BL/6 mice. Melatonin treatment significantly increased the total length and complexity of dendrites in the apical and basal cornu ammonis (CA) 1 and in the dentate gyrus in mouse hippocampi. Spine density in CA1 apical dendrites was increased, but no significant differences in other subregions were observed. In primary cultured hippocampal neurons, the length and arborization of neurites were significantly augmented by melatonin treatment. Additionally, western blot and immunohistochemical analyses in both in vivo and in vitro systems revealed significant increases in the level of cysteine-rich protein 1 (crp-1) protein, which is known to be involved in dendritic branching in mouse hippocampal neurons after melatonin treatment. Our results suggest that exogenous melatonin leads to significant alterations of neuronal micromorphometry in the adult hippocampus, possibly via crp-1 signaling.

Epigenome-wide DNA methylation analysis of small cell lung cancer cell lines suggests potential chemotherapy targets.

BACKGROUND: Small cell lung cancer (SCLC) is an aggressive neuroendocrine lung cancer. SCLC progression and treatment resistance involve epigenetic processes. However, links between SCLC DNA methylation and drug response remain unclear. We performed an epigenome-wide study of 66 human SCLC cell lines using the Illumina Infinium MethylationEPIC BeadChip array. Correlations of SCLC DNA methylation and gene expression with in vitro response to 526 antitumor agents were examined. RESULTS: We found multiple significant correlations between DNA methylation and chemosensitivity. A potentially important association was observed for TREX1, which encodes the 3' exonuclease I that serves as a STING antagonist in the regulation of a cytosolic DNA-sensing pathway. Increased methylation and low expression of TREX1 were associated with the sensitivity to Aurora kinase inhibitors AZD-1152, SCH-1473759, SNS-314, and TAK-901; the CDK inhibitor R-547; the Vertex ATR inhibitor Cpd 45; and the mitotic spindle disruptor vinorelbine. Compared with cell lines of other cancer types, TREX1 had low mRNA expression and increased upstream region methylation in SCLC, suggesting a possible relationship with SCLC sensitivity to Aurora kinase inhibitors. We also identified multiple additional correlations indicative of potential mechanisms of chemosensitivity. Methylation of the 3'UTR of CEP350 and MLPH, involved in centrosome machinery and microtubule tracking, respectively, was associated with response to Aurora kinase inhibitors and other agents. EPAS1 methylation was associated with response to Aurora kinase inhibitors, a PLK-1 inhibitor and a Bcl-2 inhibitor. KDM1A methylation was associated with PLK-1 inhibitors and a KSP inhibitor. Increased promoter methylation of SLFN11 was correlated with resistance to DNA damaging agents, as a result of low or no SLFN11 expression. The 5' UTR of the epigenetic modifier EZH2 was associated with response to Aurora kinase inhibitors and a FGFR inhibitor. Methylation and expression of YAP1 were correlated with response to an mTOR inhibitor. Among non-neuroendocrine markers, EPHA2 was associated with response to Aurora kinase inhibitors and a PLK-1 inhibitor and CD151 with Bcl-2 inhibitors. CONCLUSIONS: Multiple associations indicate potential epigenetic mechanisms affecting SCLC response to chemotherapy and suggest targets for combination therapies. While many correlations were not specific to SCLC lineages, several lineage markers were associated with specific agents.

Sestrin2 as a potential therapeutic target for cardiovascular diseases.

Sestrin2 is a cysteine sulfinyl reductase that plays crucial roles in regulation of antioxidant actions. Sestrin2 provides cytoprotection against multiple stress conditions, including hypoxia, endoplasmic reticulum (ER) stress and oxidative stress. Recent research reveals that upregulation of Sestrin2 is induced by various transcription factors such as p53 and activator protein 1 (AP-1), which further promotes AMP-activated protein kinase (AMPK) activation and inhibits mammalian target of rapamycin protein kinase (mTOR) signaling. Sestrin2 triggers autophagy activity to reduce cellular reactive oxygen species (ROS) levels by promoting nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2) activation and Kelch-like ECH-associated protein 1 (Keap1) degradation, which plays a pivotal role in homeostasis of metabolic regulation. Under hypoxia and ER stress conditions, elevated Sestrin2 expression maintains cellular homeostasis through regulation of antioxidant genes. Sestrin2 is responsible for diminishing cellular ROS accumulation through autophagy via AMPK activation, which displays cardioprotection effect in cardiovascular diseases. In this review, we summarize the recent understanding of molecular structure, biological roles and biochemical functions of Sestrin2, and discuss the roles and mechanisms of Sestrin2 in autophagy, hypoxia and ER stress. Understanding the precise functions and exact mechanism of Sestrin2 in cellular homeostasis will provide the evidence for future experimental research and aid in the development of novel therapeutic strategies for cardiovascular diseases.

Gene Expression Profiling of the Liver and Lung in Mice After Exposure to ZnO Quantum Dots.

INTRODUCTION: ZnO quantum dots (QDs) have drawn much attention recently as they are Cd-free, low-cost, and have excellent optical properties. With the expanded production and application of ZnO nanoparticles, concerns about their potential toxicity have also been raised. MATERIALS AND METHODS: We used RNA sequencing (RNA-seq) to analyze the global gene expression of liver and lung tissues after ZnO QDs treatment. Differentially expressed genes (DEGs) were screened, with a fold change >1.5 and padj <0.05. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes enrichment analyses were performed, and padj <0.05 was considered significantly enriched. The RNA-seq results were validated by quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: A total of 47 and 218 genes were significantly differentially expressed in the liver and lung. Eight GO terms were enriched in the liver and lung, and retinol metabolism and the peroxisome proliferator-activated receptor (PPAR) signaling pathway were shared in different tissues. DISCUSSION: According to DEGs and pathway enrichment analyses, inflammation might be induced in liver and lung tissues after intravenous injection of ZnO QDs. These findings will be helpful for future research and application of ZnO QDs.

New screening system using Twist1 promoter activity identifies dihydrorotenone as a potent drug targeting cancer-associated fibroblasts.

Cancer-associated fibroblasts (CAFs) are the most abundant stromal cells in tumor microenvironments. These cells strongly support tumor progression and are considered to be potent therapeutic targets. Therefore, drugs targeting CAFs have been developed, but most of them have failed in clinical trials. The discovery of additional drugs to inactivate or eliminate CAFs is thus essential. In this study, we developed a high-throughput screening system to find anti-CAF drugs using reporter cells that express Twist1 promoter-GFP. This screening system uses the activity of the Twist1 promoter as an indicator of CAF activation because Twist1 is known to be a central player in CAF activation. Using this screening system, we found that dihydrorotenone (DHR), an inhibitor of electron transfer chain complex 1 in mitochondria, can effectively deactivate CAFs. DHR-treated CAFs exhibited reduced expression of CAF-enriched markers, decreased capability of collagen gel contraction, and impaired ability to engage in tumor-promoting activities, such as facilitating the proliferation and colonization of cancer cells. Furthermore, conditioned media from DHR-treated CAFs attenuated tumor progression in mice grafted with MNK28 cells. In conclusion, DHR can be considered as a candidate drug targeting CAFs.

Identification of viral SIM-SUMO2-interaction inhibitors for treating primary effusion lymphoma.

Primary effusion lymphoma (PEL) is an aggressive B-cell malignancy without effective treatment, and caused by the infection of Kaposi's sarcoma-associated herpesvirus (KSHV), predominantly in its latent form. Previously we showed that the SUMO2-interacting motif within the viral latency-associated nuclear antigen (LANASIM) is essential for establishment and maintenance of KSHV latency. Here, we developed a luciferase based live-cell reporter system to screen inhibitors selectively targeting the interaction between LANASIM and SUMO2. Cambogin, a bioactive natural product isolated from the Garcinia genus (a traditional herbal medicine used for cancer treatment), was obtained from the reporter system screening to efficiently inhibit the association of SUMO2 with LANASIM, in turn reducing the viral episome DNA copy number for establishment and maintenance of KSHV latent infection at a low concentration (nM). Importantly, Cambogin treatments not only specifically inhibited proliferation of KSHV-latently infected cells in vitro, but also induced regression of PEL tumors in a xenograft mouse model. This study has identified Cambogin as a novel therapeutic agent for treating PEL as well as eliminating persistent infection of oncogenic herpesvirus.

Targeting RNA Polymerase I with Hernandonine Inhibits Ribosomal RNA Synthesis and Tumor Cell Growth.

RNA polymerase I (RNA Pol. I) activity is consistently expanded in multiplying cells to continue the expanded interest for ribosome generation and protein synthesis, which are fundamental for cell development and division. Thus, selective inhibitors of RNA Pol. I may offer a general helpful intends to block cancer cell multiplication. Hernandonine, isolated from the root wood of Hernandia nymphaeifolia, causes rearrangement of nucleolar proteins consistent with segregation of the nucleolus, a hallmark of RNA Pol. I transcription stress. Furthermore, the compound destabilizes RPA194, the large catalytic protein of RNA Pol. I, in a proteasome-dependent manner and inhibits nascent rRNA synthesis and expression of the 45S rRNA precursor. Finally, hernandonine induces cellular apoptosis through a p53-dependent or p53-independent process in solid tumor cell lines. These outcomes feature the prevailing effect of RNA Pol. I transcription stress on apoptosis pathway initiation and present a synthetically novel and significant molecule that represses RNA Pol. I, making it a potential objective for malignancy treatment. IMPLICATIONS: Our findings position hernandonine as a potential, particular, and orally administered cancer treatment agent appropriate for use in investigational clinical trials.

Molecular targets of biofabricated silver nanoparticles in Candida albicans.

We have analyzed the expressions of genes which regulate Ras-cAMP-EFG1 and CEK1-MAPK pathways involved in yeast to hyphal form morphogenesis in Candida albicans. The expression profile of genes associated with serum-induced morphogenesis showed reduced expressions of genes involved in these pathways by the treatment with biofabricated silver nanoparticles. Cell elongation gene, ECE1, was downregulated by 5.1 fold by the treatment of silver nanoparticles. Expression of hyphal inducer gene, TEC1 was downregulated by 6.28 fold. Negative regulators of yeast to hyphal transition, TUP1 and RFG1 were downregulated by 2.45 and 5.43 fold, respectively. Current study suggests that silver nanoparticles affect gene expression and may subsequently reduce virulence in C. albicans. Targeting genes involved in virulence may be an acceptable novel treatment strategy for pathogenic fungal infections.

Liuwei Dihuang pill cures postmenopausal osteoporosis with kidney-Yin deficiency: Potential therapeutic targets identified based on gene expression profiling.

This study aimed to investigate the potential therapeutic targets of Liuwei Dihuang pill (LDP) in the treatment of postmenopausal osteoporosis with kidney-Yin deficiency (PMO-KY).Gene expression data were downloaded from the GEO database, including 4 PMO-KY samples and 3 healthy postmenopausal controls from GSE56116, as well as 3 PMO-KY samples before LDP treatment and 3 PMO-KY samples after three months of LDP treatment from GSE57273. Limma package was used to identify differentially expressed genes (DEGs). Afterwards, the potential target genes of LDP (namely key DEGs) were identified according to the comparison of DEGs in PMO-KY group and the DEGs in LDP treatment groups. Subsequently, iRegulon plugin in Cytoscape software was used to predict potential transcription factors (TFs) that regulated the key DEGs, and Comparative Toxicogenomics Database was utilized to identify known PMO-related genes among the key DEGs.Totally, 202 and 2066 DEGs were identified between PMO-KY and controls, as well as after-treatment and before-treatment groups, respectively. Among them, 52 DEGs were up-regulated in PMO-KY but down-regulated after LDP treatment, and 8 TFs were predicted to these DEGs. Furthermore, 34 DEGs were down-regulated in PMO-KY but up-regulated after treatment, and 7 TFs were predicted to regulate these DEGs. Additionally, 43 of the 86 key DEGs were known PMO-related genes.NCOA3, TCF4, DUSP6, PELI2, and STX7 were predicted to be regulated by HOXA13. In the PMO-KY treatment, NCOA3, TCF4, DUSP6, PELI2, and STX7 might be the potential therapeutic targets of LDP. However, further investigation is required to confirm these genes.

Structures of REV1 UBM2 Domain Complex with Ubiquitin and with a Small-Molecule that Inhibits the REV1 UBM2-Ubiquitin Interaction.

REV1 is a DNA damage tolerance protein and encodes two ubiquitin-binding motifs (UBM1 and UBM2) that are essential for REV1 functions in cell survival under DNA-damaging stress. Here we report the first solution and X-ray crystal structures of REV1 UBM2 and its complex with ubiquitin, respectively. Furthermore, we have identified the first small-molecule compound, MLAF50, that directly binds to REV1 UBM2. In the heteronuclear single quantum coherence NMR experiments, peaks of UBM2 but not of UBM1 are significantly shifted by the addition of ubiquitin, which agrees to the observation that REV1 UBM2 but not UBM1 is required for DNA damage tolerance. REV1 UBM2 interacts with hydrophobic residues of ubiquitin such as L8 and L73. NMR data suggest that MLAF50 binds to the same residues of REV1 UBM2 that interact with ubiquitin, indicating that MLAF50 can compete with the REV1 UBM2-ubiquitin interaction orthosterically. Indeed, MLAF50 inhibited the interaction of REV1 UBM2 with ubiquitin and prevented chromatin localization of REV1 induced by cisplatin in U2OS cells. Our results structurally validate REV1 UBM2 as a target of a small-molecule inhibitor and demonstrate a new avenue to targeting ubiquitination-mediated protein interactions with a chemical tool.

Genistein sensitizes glioblastoma cells to carbon ions via inhibiting DNA-PKcs phosphorylation and subsequently repressing NHEJ and delaying HR repair pathways.

BACKGROUND AND PURPOSE: Previously, we found genistein could sensitize cancer cells to low linear energy transfer (LET) X-rays via inhibiting DNA-PKcs activities. Especially, high-LET heavy ion produces more DNA double strand breaks (DSBs) than low-LET radiation. Thus, the study was designed to investigate the detailed molecular mechanisms of genistein on sensitizing cancer cells to heavy ions. MATERIALS AND METHODS: Human glioblastoma (GBM) cell lines with or without genistein pre-treatment were irradiated with high-LET carbon ions. Cell survival was determined with colony formation assay. DNA DSBs were evaluated by means of detecting γ-H2AX foci and immuno-blotting DSB repair proteins, cell apoptosis was detected using Annexin V and PI staining. The interaction of genistein with DNA-PKcs activation site was estimated by molecular docking in the autodock software. RESULTS: Genistein sensitized DNA-PKcs proficient GBM cells to high-LET carbon ions via delaying the clearance of γ-H2AX foci. Genistein was physically bound to DNA-PKcs and functionally inhibited the phosphorylation of DNA-PKcs. Consequently, the non-homologous end joining (NHEJ) repair of DSBs was inhibited and the homologous recombination (HR) repair was delayed by genistein, thereby leading to an increase in apoptosis in DNA-PKcs proficient GBM cells after irradiation. CONCLUSION: Our study demonstrated that genistein holds promise as a radiosensitizer for enhancing the efficacy of carbon ion radiotherapy against DNA-PKcs proficient GBM via inhibiting DNA-PKcs phosphorylation and subsequently repressing NHEJ and delaying HR repair pathways.

Reducing Mutant Huntingtin Protein Expression in Living Cells by a Newly Identified RNA CAG Binder.

Expanded CAG trinucleotide repeats in Huntington's disease (HD) are causative for neurotoxicity. The mutant CAG repeat RNA encodes neurotoxic polyglutamine proteins and can lead to a toxic gain of function by aberrantly recruiting RNA-binding proteins. One of these is the MID1 protein, which induces aberrant Huntingtin (HTT) protein translation upon binding. Here we have identified a set of CAG repeat binder candidates by in silico methods. One of those, furamidine, reduces the level of binding of HTT mRNA to MID1 and other target proteins in vitro. Metadynamics calculations, fairly consistent with experimental data measured here, provide hints about the binding mode of the ligand. Importantly, furamidine also decreases the protein level of HTT in a HD cell line model. This shows that small molecules masking RNA-MID1 interactions may be active against mutant HTT protein in living cells.

Dynamic Changes in the Protein Localization in the Nuclear Environment in Pancreatic ß-Cell after Brief Glucose Stimulation.

Characterization of molecular mechanisms underlying pancreatic ß-cell function in relation to glucose-stimulated insulin secretion is incomplete, especially with respect to global response in the nuclear environment. We focus on the characterization of proteins in the nuclear environment of ß-cells after brief, high glucose stimulation. We compared purified nuclei derived from ß-cells stimulated with 17 mM glucose for 0, 2, and 5 min using quantitative proteomics, a time frame that most likely does not result in translation of new protein in the cell. Among the differentially regulated proteins, we identified 20 components of the nuclear organization processes, including nuclear pore organization, ribonucleoprotein complex, and pre-mRNA transcription. We found alteration of the nuclear pore complex, together with calcium/calmodulin-binding chaperones that facilitate protein and RNA import or export to/from the nucleus to the cytoplasm. Putative insulin mRNA transcription-associated factors were identified among the regulated proteins, and they were cross-validated by Western blotting and confocal immunofluorescence imaging. Collectively, our data suggest that protein translocation between the nucleus and the cytoplasm is an important process, highly involved in the initial molecular mechanism underlying glucose-stimulated insulin secretion in pancreatic ß-cells.