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Abstract: (±)-anti-Benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE), which causes bulky-adduct DNA damage, is well-characterized as the ultimate carcinogen of benzo[a]pyrene (BaP). In this study, we have employed Affymetrix HG-U133 Plus 2.0 microarray and quantitative real-time RT-PCR methods to investigate a temporal transcriptomic response triggered by a low concentration (0.05μM) of BPDE at 1, 10, and 22h after exposure in normal human cells. The differential gene expression profiles at the three time points varied greatly, and generally reflected a cellular responsive process from initiation to progression and to recovery after the BPDE-caused damage. The dynamic regulation of the genes related with cell cycle progression and cell fate exhibited a tendency from inhibition to survival, which was accordant with the cell cycle arrest and cytotoxicity data induced by the low-dose BPDE exposure. In silico comparison of the genomic data revealed that BPDE and ultraviolet induced a panel of common transcriptional responses, which might be related with a series of similar molecular processes elicited by these two DNA-damaging agents. In conclusion, this whole-genome time-course study has identified a dynamically regulated transcriptional signature after low-dose BPDE exposure, which may help to understand the complex mechanisms of mutagenesis and carcinogenesis induced by BPDE. [Copyright &y& Elsevier]

Abstract: (±)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE) is a carcinogen causing bulky-adduct DNA damage. In this study, we investigated early transcriptional signatures induced by various concentrations (0.005, 0.05, and 0.5 μM) of this carcinogen in a normal human cell line (FL human amnion epithelial cells) using the whole-genome Affymetrix HG-U133 Set microarray. The numerous identified genes were involved in multiple functions and higher doses of BPDE elicited more robust expression changes. The disturbance of genes involved in cell cycle regulation, growth and apoptosis was correlated with the S and G2/M phase cell cycle arrest and cytotoxic phenotypes induced by different levels of BPDE. Bioinformatic analysis showed that several transcription factors and their related stress signaling pathways might partly account for the transcriptional signature induced by BPDE. Additionally, gene ontology analysis of the microarray data showed down-regulation of transport, cytoskeleton and DNA repair by 0.5 μM BPDE exposure. In conclusion, this genomic analysis helps to understand the mechanism of cellular response to BPDE. [Copyright &y& Elsevier]

Abstract: The alkylating agent N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) induces cellular DNA damage and other comprehensive alterations that lead to chromosomal aberrations, mutations, tumor initiation, and cell death. However, the molecular mechanism of MNNG-induced cellular stress remains unclear. We undertook a genome-wide analysis of early transcriptional responses of human FL amnion epithelial cells to three relatively low doses of MNNG (0.2, 1.0, and 10.0μM). Using Affymetrix GeneChip HG-U133 Set oligonucleotide microarrays, a total of 281 genes were found to change their expression after exposure to all three doses of MNNG compared with the vehicle control, DMSO. Of these, 70, 112, and 146 genes showed different expression levels following treatment with low, medium, and high-dose MNNG, respectively. A subset of these genes were selected for further confirmation using quantitative real-time RT-PCR with ABI TaqMan® low-density arrays, and the differential expression of 33 genes was validated. The results demonstrate that low doses of MNNG can induce various changes in gene expression at an early stage of exposure. The responsive genes are involved in multiple cellular biological processes including transcription regulation, signal transduction, cell cycle regulation, cytoskeleton organization, protein synthesis, immune responses, and metabolism. The cell cycle progression was down-regulated, in which several genes were validated involved, including the cell cycle regulators (CDK6, STK6, CENPA, and CCNF), the transcription factor ID-1, and the calcium signaling molecules (CAMK2G and NFAT5). The possible roles of the responsive genes and their related pathways in MNNG-induced cellular responses are discussed. This study helps to complete the picture of how cells respond to environmental chemical exposure via transcriptional regulation. [Copyright &y& Elsevier]

Abstract: Ribonucleotide reductase (RR) is an important therapeutic target for anticancer drugs. The structure of human RR features a 1:1 complex of two homodimeric subunits, hRRM1 and hRRM2. p53R2 is a newly identified homologue of hRRM2. We have devised a holoenzyme-based in vitro assay for the determination of the potency and subunit-selectivity of small-molecule inhibitors of RR. The assay was implemented using two forms of recombinant RR (hRRM2/hRRM1 and p53R2/hRRM1) and based on their [3H]CDP reduction activity. Hydroxyurea was used to standardize the assay. We found that the activities of hRRM2/hRRM1 and p53R2/hRRM1 were decreased by hydroxyurea in a dose-dependent manner. The –NH–OH segment of hydroxyurea was shown to be essential for inhibition. In the presence of Fe(III) and reductants, less inhibition of enzymatic activity by hydroxyurea was observed, especially for p53R2/hRRM1. The potency of four hydroxyurea analogues (Schiff bases of hydroxysemicarbazide, SB-HSC) decreased in the order SB-HSC 21>SB-HSC 24>SB-HSC 2>hydroxyurea (HU)>SB-HSC 29. SB-HSC 2 and SB-HSC 24 inhibited p53R2/hRRM1 significantly more than hRRM2/hRRM1, whereas SB-HSC 21 and SB-HSC 29 showed low subunit-selectivity. Electron paramagnetic resonance (EPR) measurements showed that inhibition of RR was accompanied by reduction of its tyrosyl radical. The method was validated by comparison with data obtained using cell-based assays. We suggest that this novel recombinant-holoenzyme-based in vitro assay is a useful tool for the discovery of more potent and subunit-selective inhibitors of RR. [Copyright &y& Elsevier]

A sensitive surface enhanced Raman spectroscopy (SERS) substrate based on SiO2@Ag core-shell with a QuEChERs pretreatment method were constructed for the determination of carbendazim residue in tobacco leaves. The SERS substrate was formed by self-assembly of monolayer homogeneous SiO2 nanospheres onto silicon wafer following by vacuum thermal evaporation of silver film. The QuEChERs pretreatment method was used to eliminate the interfering effects of pigments and organic acids in tobacco leaves. The results for carbendazim in tobacco leaves show the limit of detection (LOD) of the SERS substrate was 0.1 mg/kg and the recovery rate was from 82.56% to 95.93%. The proposed low-cost sensitive SERS substrates provide potential application for tobacco safety monitoring. [ABSTRACT FROM AUTHOR]

Activation of the Toll-like receptor 4 (TLR4) by bacterial endotoxins in macrophages plays a crucial role in the pathogenesis of sepsis. However, the mechanism underlying TLR4 activation in macrophages is still not fully understood. Here, we reveal that upon lipopolysaccharide (LPS) stimulation, lysine acetyltransferase CBP is recruited to the TLR4 signalosome complex leading to increased acetylation of the TIR domains of the TLR4 signalosome. Acetylation of the TLR4 signalosome TIR domains significantly enhances signaling activation via NF-κB rather than IRF3 pathways. Induction of NF-κB signaling is responsible for gene expression changes leading to M1 macrophage polarization. In sepsis patients, significantly elevated TLR4-TIR acetylation is observed in CD16+ monocytes combined with elevated expression of M1 macrophage markers. Pharmacological inhibition of HDAC1, which deacetylates the TIR domains, or CBP play opposite roles in sepsis. Our findings highlight the important role of TLR4-TIR domain acetylation in the regulation of the immune responses in sepsis, and we propose this reversible acetylation of TLR4 signalosomes as a potential therapeutic target for M1 macrophages during the progression of sepsis. [ABSTRACT FROM AUTHOR]

Surface-enhanced Raman spectroscopy (SERS) has great potential for rapid detection of trace levels of pesticides in food. Scientists have much interested in this analytical technique due to its sensitivity, rapid detection, simplicity, and low cost. However, most of researchers are only focusing on the detection of a single pesticide residue. In this paper, we firstly report the simultaneous detection of two pesticide residues in tobacco leaves by using composite SERS substrate of SiO 2 @Ag nanoparticles. The entire analysis time is less than 10 min. The detection results show satisfactory linearity in the range of 0.1–25 mg/kg and 1–25 mg/kg with the limit of detection of 0.1 mg/kg and 1 mg/kg for carbendazim and flumetralin in tobacco samples, respectively. The recovery rates of carbendazim and flumetralin in the tobacco leaves are 85.2 ∼ 112.12 % and 90.38 ∼ 113.42 %, respectively. The detection results also show good uniformity and reproducibility, with a relative standard deviation(RSD) value of 4.4 % between substrates. Our reported substrate combined with portable Raman spectrometer provides a powerful tool for simultaneous detection of multiple pesticide residues for food safety. • Simultaneous detection of mixed pesticide residues in tobacco using SERS method. • A portable Raman spectrometer was used for rapid detection of pesticide residues. • The compact islandshaped silver nanoparticles provide an abundance of "hot spots". • The QuEChERs method was used to effectively remove impurities from tobacco leaves. [ABSTRACT FROM AUTHOR]

Simple Summary: The clinical treatment of acute myeloid leukaemia is still dominated by chemotherapy. Clinically used anti-leukaemia drugs have shortcomings such as myelosuppression, toxicity and drug resistance. Therefore, the need to develop other chemotherapeutic drugs to meet more clinical needs is urgent. Ribonucleotide reductase (RNR) consists of a catalytic large subunit M1 (RRM1) and a regulatory small subunit M2 (RRM2), which provides dNTPs for DNA synthesis. The rapid proliferation of cancer cells requires large amounts of dNTPs. Therefore, the use of RNR inhibitors is a promising strategy for the clinical treatment of various malignancies. Monobenzone is an FDA-approved depigmenting agent for vitiligo patients. In this study, we demonstrate that monobenzone is a potent inhibitor of RNR enzyme activity by targeting RRM2 protein, and thus has significant anti-leukaemia efficacy in vitro and in vivo. This finding suggests that monobenzone has the potential to be optimized as a novel anti-AML therapeutic drug in the future. Acute myeloid leukaemia (AML) is one of the most common types of haematopoietic malignancy. Ribonucleotide reductase (RNR) is a key enzyme required for DNA synthesis and cell proliferation, and its small subunit RRM2 plays a key role for the enzymatic activity. We predicted monobenzone (MB) as a potential RRM2 target compound based on the crystal structure of RRM2. In vitro, MB inhibited recombinant RNR activity (IC50 = 0.25 μM). Microscale thermophoresis indicated that MB inhibited RNR activity by binding to RRM2. MB inhibited cell proliferation (MTT IC50 = 6–18 μM) and caused dose-dependent DNA synthesis inhibition, cell cycle arrest, and apoptosis in AML cells. The cell cycle arrest was reversed by the addition of deoxyribonucleoside triphosphates precursors, suggesting that RNR was the intracellular target of the compound. Moreover, MB overcame drug resistance to the common AML drugs cytarabine and doxorubicin, and treatment with the combination of MB and the Bcl-2 inhibitor ABT-737 exerted a synergistic inhibitory effect. Finally, the nude mice xenografts study indicated that MB administration produced a significant inhibitory effect on AML growth with relatively weak toxicity. Thus, we propose that MB has the potential as a novel anti-AML therapeutic agent in the future. [ABSTRACT FROM AUTHOR]

A rapid method for determination of parabens preservatives (methyl paraben, ethyl paraben, isopropyl paraben, propyl paraben, isobutyl paraben, and butyl paraben) in flavors was established by using supercritical fluid chromatography‐tandem mass spectrometry combined with dispersive solid‐phase extraction. After adding methanol and primary secondary amine to the sample simultaneously, high extraction efficiency and good sample cleanup could be obtained by simple shaking. Parabens were well separated on a Chiralpak IG‐3 column in 6 min by gradient elution. Recoveries from spiked blank samples at 0.5, 1.0, and 5.0 mg/kg were determined to be 88.3–106.6%with relative standard deviations less than 8.0%. All analytes achieved good linear relation (r ≥ 0.999 2). The limits of detection for all analytes ranged from 0.03 to 0.09 mg/kg and the limits of quantification from 0.11 to 0.31 mg/kg, respectively. A total of 20 actual samples were successfully analyzed by taking the proposed method. Being simple, rapid, green, and reliable, this method can be taken for the determination of parabens preservatives in flavors. [ABSTRACT FROM AUTHOR]

Background: Aberrant DNA repair pathways contribute to malignant transformation or disease progression and the acquisition of drug resistance in multiple myeloma (MM); therefore, these pathways could be therapeutically exploited. Ribonucleotide reductase (RNR) is the rate-limiting enzyme for the biosynthesis of deoxyribonucleotides (dNTPs), which are essential for DNA replication and DNA damage repair. In this study, we explored the efficacy of the novel RNR inhibitor, 4-hydroxysalicylanilide (HDS), in myeloma cells and xenograft model. In addition, we assessed the clinical activity and safety of HDS in patients with MM. Methods: We applied bioinformatic, genetic, and pharmacological approaches to demonstrate that HDS was an RNR inhibitor that directly bound to RNR subunit M2 (RRM2). The activity of HDS alone or in synergy with standard treatments was evaluated in vitro and in vivo. We also initiated a phase I clinical trial of single-agent HDS in MM patients (ClinicalTrials.gov: NCT03670173) to assess safety and efficacy. Results: HDS inhibited the activity of RNR by directly targeting RRM2. HDS decreased the RNR-mediated dNTP synthesis and concomitantly inhibited DNA damage repair, resulting in the accumulation of endogenous unrepaired DNA double-strand breaks (DSBs), thus inhibiting MM cell proliferation and inducing apoptosis. Moreover, HDS overcame the protective effects of IL-6, IGF-1 and bone marrow stromal cells (BMSCs) on MM cells. HDS prolonged survival in a MM xenograft model and induced synergistic anti-myeloma activity in combination with melphalan and bortezomib. HDS also showed a favorable safety profile and demonstrated clinical activity against MM. Conclusions: Our study provides a rationale for the clinical evaluation of HDS as an anti-myeloma agent, either alone or in combination with standard treatments for MM. Trial registration: ClinicalTrials.gov, NCT03670173, Registered 12 September 2018. [ABSTRACT FROM AUTHOR]

Background: Aberrant DNA repair pathways contribute to malignant transformation or disease progression and the acquisition of drug resistance in multiple myeloma (MM); therefore, these pathways could be therapeutically exploited. Ribonucleotide reductase (RNR) is the rate-limiting enzyme for the biosynthesis of deoxyribonucleotides (dNTPs), which are essential for DNA replication and DNA damage repair. In this study, we explored the efficacy of the novel RNR inhibitor, 4-hydroxysalicylanilide (HDS), in myeloma cells and xenograft model. In addition, we assessed the clinical activity and safety of HDS in patients with MM. Methods: We applied bioinformatic, genetic, and pharmacological approaches to demonstrate that HDS was an RNR inhibitor that directly bound to RNR subunit M2 (RRM2). The activity of HDS alone or in synergy with standard treatments was evaluated in vitro and in vivo. We also initiated a phase I clinical trial of single-agent HDS in MM patients (ClinicalTrials.gov: NCT03670173) to assess safety and efficacy. Results: HDS inhibited the activity of RNR by directly targeting RRM2. HDS decreased the RNR-mediated dNTP synthesis and concomitantly inhibited DNA damage repair, resulting in the accumulation of endogenous unrepaired DNA double-strand breaks (DSBs), thus inhibiting MM cell proliferation and inducing apoptosis. Moreover, HDS overcame the protective effects of IL-6, IGF-1 and bone marrow stromal cells (BMSCs) on MM cells. HDS prolonged survival in a MM xenograft model and induced synergistic anti-myeloma activity in combination with melphalan and bortezomib. HDS also showed a favorable safety profile and demonstrated clinical activity against MM. Conclusions: Our study provides a rationale for the clinical evaluation of HDS as an anti-myeloma agent, either alone or in combination with standard treatments for MM. Trial registration: ClinicalTrials.gov, NCT03670173, Registered 12 September 2018. [ABSTRACT FROM AUTHOR]

A novel method has been developed for the simultaneous determination of multiclass pesticide residues in tobacco and cigarette smoke, using a modified QuEChERS (quick, easy, cheap, effective, rugged and safe) procedure and ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS). Cigarette mainstream smoke particulate was collected on a Cambridge filter pad. Pesticide residues was extracted with an aqueous solution, back extracted into acetonitrile after freezing, purified by dispersive solid phase extraction with primary-secondary amine adsorbents and analyzed by UPLC–MS/MS. The obtained mean recoveries of 16 pesticides commonly used on tobacco at three fortification levels (5.9, 94.1 and 352.9 ng g−1) ranged from 69.3 to 115.9% with relative standard deviations between 2.4 and 11.3%. The limits of detection ranged from 0.14 to 13.28 ng g−1. Finally, the proposed method was applied to study the pesticide smoke transfer ratio in 2 cigarettes with pesticide standard spiked and 51 cigarettes with one or more pesticide residues. The transfer ratio of pesticides residue in tobacco into the smoke might be much less than that from artificially spiked tobacco (<25%) with spiking levels varied from 1.88 to 9.41 μg g−1. The transfer ratio of pesticide from artificially spiked tobacco into cigarette mainstream smoke was from 0.0 to 56.5%, and pesticide residues from tobacco into cigarette smoke were from 0.0 to 26.1% using the ISO smoking method (ISO 3308 2012). [ABSTRACT FROM AUTHOR]

Aims: Alternaria longipes is a causal agent of brown spot of tobacco, which remains a serious threat to tobacco production. Herein, we established a detection method for A. longipes in tobacco samples based on the principle of time‐resolved fluoroimmunoassay, in order to fulfil the requirement of rapid, sensitive and accurate detection in situ. Methods and Results: A monoclonal antibody against A. longipes was generated, and its purity and titration were assessed using western blot and ELISA. The size of europium (III) nanospheres was measured to confirm successful antibody conjugation. The method described here can detect A. longipes protein lysates as low as 0.78 ng ml−1, with recovery rates ranging from 85.96% to 99.67% in spiked tobacco. The specificity was also confirmed using a panel of microorganisms. Conclusions: The fluorescent strips allow rapid and sensitive onsite detection of A. longipes in tobacco samples, with high accuracy, specificity, and repeatability. Significance and Impact of the Study: This novel detection method provides convenience of using crude samples without complex procedures, and therefore allows rapid onsite detection by end users and quick responses towards A. longipes, which is critical for disease control and elimination of phytopathogens. [ABSTRACT FROM AUTHOR]

Liver ageing not only damages liver function but also harms systemic metabolism. To better understand the mechanisms underlying liver ageing, we transplanted the livers of young rats to young and old rats and performed untargeted metabolomics to detect changes in the metabolites in the liver tissues and sera. A total of 153 metabolites in the livers and 83 metabolites in the sera were different between the old and young rats that did not undergo liver transplantation; among these metabolites, 7 different metabolites were observed in both the livers and sera. Five weeks after liver transplantation, the levels of 25 metabolites in the young donor livers were similar to those in the old rats, and this result probably occurred due to the effect of the whole‐body environment of the older recipients on the young livers. The 25 altered metabolites included organic acids and derivatives, lipids and lipid‐like molecules, etc. In the sera, the differences in 78 metabolites, which were significant between the young and old rats, were insignificant in the old recipient rats and made the metabolic profile of the old recipients more similar to that of the young recipients. Finally, combining the above metabolomic data with the transcriptomic data from the GEO, we found that the altered metabolites and genes in the liver were enriched in 9 metabolic pathways, including glycerophospholipid, arachidonic acid, histidine and linoleate. Thus, this study revealed important age‐related metabolites and potential pathways as well as the interaction between the liver and the whole‐body environment. [ABSTRACT FROM AUTHOR]

• The stereoselective separation of the triazole fungicides is achieved on an ACQUITY UPC2 Trefoil AMY 1 column in 6 min. • The rapid PRiME pass-through cleanup combined with SFC-MS/Ms is used to ensure high efficiency stereoselective separation. • The matrix effects of different isomers are very different. • The proposed method shows a good performance in the precision, linearity, selectivity, and sensitivity for all studied stereoisomers in tobacco. This work presents a simple, rapid and green chiral analysis method for five triazole fungicides (penconazole, tebuconazole, triadimefon, myclobutanil, and triadimenol) in tobacco, by which the samples were cleaned up by the novel pass-through solid phase extraction and subsequently the stereoisomers were separated and determined by the supercritical fluid chromatography-tandem mass spectrometry (SFC-MS/MS). Optimized separation of the stereoisomers was achieved on an ACQUITY UPC2 Trefoil AMY 1 column within 6 min. Under fortified concentration levels of 0.1, 0.5 and 2.0 mg/kg, the mean recoveries were 82.8-106.6%, the intra-day relative standard deviations (RSDs) were 1.1-6.6%, and the inter-day RSDs were 2.5-5.6%. The correlation coefficient was greater than 0.9926 for all studied analytes within the range of 10-500 ng/mL. The limits of detection (LODs) for all stereoisomers ranged from 0.26 μg/kg to 3.24 μg/kg. The established method was subsequently successfully applied to analyze authentic samples, confirming that this method is a novel, rapid and environmentally friendly method for the stereoselective separation of triazole fungicides in tobacco. [ABSTRACT FROM AUTHOR]

Purpose: Esophageal cancer (EC) is an aggressive malignancy and is often resistant to currently available therapies. Inhibition of ribonucleotide reductase small subunit M2 (RRM2) in tumors is speculated to mediate chemosensitization. Previous studies have reported that Osalmid could act as an RRM2 inhibitor. We explored whether RRM2 was involved in radioresistance and the antitumor effects of Osalmid in EC.Methods and Materials: RRM2 expression was detected by immunohistochemistry in EC tissues. The effects of Osalmid on cell proliferation, apoptosis, and cell cycle were assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphhenyl tetrazolium, colony formation, and flow cytometry assays. DNA damage, cell apoptosis, and senescence induced by Osalmid or ionizing radiation (IR) alone, or both, were detected with immunofluorescence, flow cytometry, Western blot, and β-galactosidase staining. A xenograft mouse model of EC was used to investigate the potential synergistic effects of Osalmid and IR in vivo.Results: The expression of RRM2 in treatment-resistant EC tissues is much higher than in treatment-sensitive EC, and strong staining of RRM2 was correlated with shorter overall survival. We observed direct cytotoxicity of Osalmid in EC cells. Osalmid also produced inhibition of the ERK1/2 signal transduction pathway and substantially enhanced IR-induced DNA damage, apoptosis, and senescence. Furthermore, treatment with Osalmid and IR significantly suppressed tumor growth in xenograft EC models without additional toxicity to the hematologic system and internal organs.Conclusions: Our study revealed that RRM2 played a vital role in radioresistance in EC, and Osalmid synergized with IR to exert its antitumor effects both in vitro and in vivo. [ABSTRACT FROM AUTHOR]

E2F8 promotes LUAD development by transcriptionally activating RRM2 expression and E2F8 knockdown and WEE1 suppression have a synergistic inhibitory effect on LUAD by targeting both RRM2 expression and degradation pathways. [Display omitted] Ribonucleotide reductase (RR) is a rate-limiting enzyme that facilitates DNA replication and repair by reducing nucleotide diphosphates (NDPs) to deoxyribonucleotide diphosphates (dNDPs) and is thereby crucial for cell proliferation and cancer development. The E2F family of transcription factors includes key regulators of gene expression involved in cell cycle control. In this study, E2F8 expression was significantly increased in most cancer tissues of lung adenocarcinoma (LUAD) patients and was correlated with the expression of RRM2 through database and clinical samples analysis. The protein expression of E2F8 and RRM2 were positively correlated with tumor–node–metastasis (TNM) pathological stage, and high expression of E2F8 and RRM2 predicted a low 5-year overall survival rate in LUAD patients. Overexpression and knockdown experiments showed that E2F8 was essential for LUAD cell proliferation, DNA synthesis, and cell cycle progression, which were RRM2-dependent. Reporter gene, ChIP-qPCR, and DNA pulldown–Western blot assays indicated that E2F8 activated the transcription of the RRM2 gene by directly binding with the RRM2 promoter in LUAD cells. Previous studies indicated that inhibition of WEE1 kinase can suppress the phosphorylation of CDK1/2 and promote the degradation of RRM2. We further showed here that the combination of E2F8 knockdown with MK-1775, an inhibitor of WEE1 being evaluated in clinical trials, synergistically suppressed proliferation and promoted apoptosis of LUAD cells in vitro and in vivo. Thus, this study reveals a novel role of E2F8 as a proto-oncogenic transcription activator by activating RRM2 expression in LUAD, and targeting both the transcription and degradation mechanisms of RRM2 could produce a synergistic inhibitory effect for LUAD treatment in addition to conventional inhibition of RR enzyme activity. [ABSTRACT FROM AUTHOR]

Liver metastases develop in more than half of the patients with colorectal cancer (CRC) and are associated with a poor prognosis. The factors influencing liver metastasis of CRC are poorly characterized, but this information is urgently needed. We have now discovered that small extracellular vesicles (sEVs; exosomes) derived from CRC can be specifically targeted to liver tissue and induce liver macrophage polarization toward an interleukin-6 (IL-6)-secreting proinflammatory phenotype. More importantly, we found that microRNA-21-5p (miR-21) was highly enriched in CRC-derived sEVs and was essential for creating a liver proinflammatory phenotype and liver metastasis of CRC. Silencing either miR-21 in CRC-sEVs or Toll-like receptor 7 (TLR7) in macrophages, to which miR-21 binds, abolished CRC-sEVs' induction of proinflammatory macrophages. Furthermore, miR-21 expression in plasma-derived sEVs was positively correlated with liver metastasis in CRC patients. Collectively, our data demonstrate a pivotal role of CRC-sEVs in promoting liver metastasis by inducing an inflammatory premetastatic niche through the miR-21–TLR7–IL-6 axis. Thus, sEVs–miR-21 represents a potential prognostic marker and therapeutic target for CRC patients with liver metastasis. [ABSTRACT FROM AUTHOR]

Background N -nitroso compounds (NOC) can cause cancers in a wide variety of animal species, and many of them are also potential human carcinogens. However, their underlying genotoxic mechanisms occurred within the context of chromatin, such as aberrant histone modifications, remained elusive. Methods We investigated the dynamic landscapes of histone modifications after N -nitroso compound N -methyl- N ′-nitro- N -nitrosoguanidine (MNNG) and N -methyl- N -nitroso-urea (MNU) exposure. Among the altered histone modifications, we also investigated the control mechanisms of histone H3 phosphorylation changes and its possible implications on transcriptional repression. Results Significantly, we find a specific biphasic reduction of histone H3 phosphorylation at serine 10 (H3S10ph) and serine 28 (H3S28ph), and a rapid decrease of histone H4 acetylation upon MNNG and MNU exposure. Further investigations reveal that the first hypophosphorylation of H3 occurs in a poly(ADP-ribosyl)ation enzyme PARP-1 (Poly(ADP-Ribose) Polymerase 1) dependent manner, whereas the second decline of H3 phosphorylation is at least partially under the control of histone kinase VRK1 (vaccinia-related kinase 1) and dependent on the tumor suppressor protein p53. In addition, DNA damage induced down-regulation of H3S10/S28 phosphorylation also functions in transcriptional repression of genes, such as cell-cycle regulators. Conclusions Alkylating damage induced by NOC elicits a biphasic reduction of histone H3 phosphorylation with distinct control mechanisms, which is contributing to DNA damage responses such as the repair-facilitated transcriptional repression. General significance Identification of the dynamic changes and underlying mechanisms of histone modifications upon NOC exposure would be of great help in understanding the epigenetic regulations of NOC induced DNA damage responses. [ABSTRACT FROM AUTHOR]

Understanding molecular mechanisms of colorectal cancer (CRC) metastasis is urgently required for targeted therapy and prognosis of metastatic CRC. In this study, we explored potential effects of silent mating type information regulation 2 homolog 1 (SIRT1) on CRC metastasis. Our data showed that ectopic expression of SIRT1 markedly increased the migration and invasion of CRC cells. In contrast, silencing SIRT1 repressed this behavior in aggressive CRC cells. Tumor xenograft experiments revealed that knockdown of SIRT1 impaired CRC metastasis in vivo. Silencing SIRT1 in CRC cells induced mesenchymal-epithelial transition (MET), which is the reverse process of epithelial-mesenchymal transition (EMT) and characterized by a gain of epithelial and loss of mesenchymal markers. We provided a mechanistic insight toward regulation of Fra-1 by SIRT1 and demonstrated a direct link between the SIRT1-Fra-1 axis and EMT. Moreover, SIRT1 expression correlated positively with Fra-1 expression, metastasis and overall survival in patients with CRC. Taken together, our data provide a novel mechanistic role of SIRT1 in CRC metastasis, suggesting that SIRT1 may serve as a potential therapeutic target for metastatic CRC. [ABSTRACT FROM AUTHOR]

Chronic hepatitis B virus (HBV) infection is a key factor for hepatocellular carcinoma worldwide. Ribonucleotide reductase (RR) regulates the deoxyribonucleoside triphosphates biosynthesis and serves as a target for anti-cancer therapy. Here, we demonstrate that RR is essential for HBV replication and the viral covalently-closed-circular DNA (cccDNA) synthesis in host liver cells. By performing computer-assisted virtual screening against the crystal structure of RR small subunit M2 (RRM2), osalmid, was identified as a potential RRM2-targeting compound. Osalmid was shown to be 10-fold more active in inhibiting RR activity than hydroxyurea, and significantly inhibited HBV DNA and cccDNA synthesis in HepG2.2.15 cells. In contrast, hydroxyurea and the RR large subunit (RRM1)-inhibitory drug gemcitabine showed little selective activity against HBV replication. In addition, osalmid also was shown to possess potent activity against a 3TC-resistant HBV strain, suggesting utility in treating drug-resistant HBV infections. Interestingly, osalmid showed synergistic effects with lamivudine (3TC) in vitro and in vivo without significant toxicity, and was shown to inhibit RR activity in vivo , thus verifying its in vivo function . Furthermore, 4-cyclopropyl-2-fluoro- N -(4-hydroxyphenyl) benzamide (YZ51), a novel derivative of osalmid, showed higher efficacy than osalmid with more potent RR inhibitory activity. These results suggest that RRM2 might be targeted for HBV inhibition, and the RRM2-targeting compound osalmid and its derivative YZ51 could be a novel class of anti-HBV candidates with potential use for hepatitis B and HBV-related HCC treatment. [ABSTRACT FROM AUTHOR]

JMJD5 is a Jumonji C domain-containing demethylase/hydroxylase shown to be essential in embryological development, osteoclastic maturation, circadian rhythm regulation and cancer metabolism. However, its role and underlying mechanisms in oncogenesis remain unclear. Here, we demonstrate that JMJD5 forms complex with the tumor suppressor p53 by interacting with p53 DNA-binding domain (DBD), and negatively regulates its activity. Downregulation of JMJD5 resulted in increased expression of multiple p53 downstream genes, such as the cell cycle inhibitor CDKN1A and DNA repair effector P53R2 , only in p53-proficient lung cancer cells. Upon DNA damage, the JMJD5–p53 association decreased, and thereby, promoted p53 recruitment to the target genes and stimulated its transcriptional activity. Furthermore, JMJD5 facilitated the cell cycle progression in a p53-dependent manner under both normal and DNA damage conditions. Depletion of JMJD5 inhibited cell proliferation and enhanced adriamycin-induced cell growth suppression in the presence of p53. Collectively, our results reveal that JMJD5 is a novel binding partner of p53 and it functions as a positive modulator of cell cycle and cell proliferation mainly through the repression of p53 pathway. Our study extends the mechanistic understanding of JMJD5 function in cancer development and implicates JMJD5 as a potential therapeutic target for cancer. [ABSTRACT FROM AUTHOR]

Abstract: JMJD5 has recently been reported to participate in circadian rhythm regulation, embryological development, osteoclastogenesis and tumorigenesis. Although JMJD5 has been found mainly localized in the nucleus of cells, how it enters the nucleus remains unclear. Here we report that JMJD5 contains a functional bipartite nuclear localization signal (NLS) and a chromosome region maintenance 1 (CRM1)-dependent nuclear export signal (NES). Importin α/β and transportin-1 were further identified as JMJD5-associated transport proteins, and different binding regions were determined for the two nuclear import receptors. Additionally, we demonstrate that both the active NLS and the JmjC domain of JMJD5 are necessary for cyclin A1 transcription. Chromatin immunoprecipitation (ChIP) analysis confirmed the alterations of di-methylated lysine 36 of histone H3 (H3K36me2) in the coding region of cyclin A1. These results reveal that the N-terminal domain is essential for the nuclear localization of JMJD5 and its normal enzymatic function towards substrates in the nucleus. [Copyright &y& Elsevier]

Abstract: DNA polymerase κ (Pol κ), a member of Y-family DNA polymerases, can synthesize DNA with moderate fidelity on undamaged DNAs and replicate accurately in vitro thymine glycol, 8-oxo-G and aromatic adducts such as benzo[a]pyrene diol epoxide (BPDE). However, few studies have been done on the transcriptional regulation of Pol κ. In this study, we predicted and cloned the promoter region of the human POLK gene. Through the analysis of deletion constructs of the POLK promoter, we demonstrated that the region −336/−141 contained repressing elements and the region −141/+226 contained positive regulatory elements for transcription of human Pol κ. Furthermore, quantitative RT-PCR showed that human POLK mRNA expression was dysregulated in FL cells treated by BPDE. The transcriptional activities of the POLK promoter regions −336/+437 and +20/+437 were significantly reduced by BPDE treatment, indicating that transcription factors in this two regions, such as HSF1, may regulate the transcription of human POLK gene in response to BPDE. [Copyright &y& Elsevier]

Abstract: Benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE) is a carcinogen causing bulky-adduct DNA damage and inducing extensive cell responses regulating cell cycle, cell survival and apoptosis. However, the mechanism of cellular responses to BPDE exposure is not fully understood. In this study, we demonstrated the involvement of the phosphorylation of the α-subunit of the eukaryotic initiation factor-2 (eIF2α) in the cellular response to BPDE exposure and addressed the role of eIF2α phosphorylation in the regulation of the cellular stress. Phosphorylation of eIF2α was induced in a normal human FL amnion epithelial cell line, and the expression of ATF4, a conserved downstream transcriptional factor of eIF2α phosphorylation, was up-regulated after BPDE exposure; however, the four known primary kinases for eIF2α phosphorylation (GCN2, HRI, PKR, and PERK) were not found activated. While BPDE induced severe cell cycle arrest and apoptosis and decreased cell viability in FL cells, salubrinal, a selective inhibitor of eIF2α dephosphorylation, maintained the eIF2α phosphorylation and attenuated cell cycle arrest and apoptosis and promoted cell survival. The findings reveal that when BPDE causes cellular damages, it induces eIF2α phosphorylation as well, which produces a pro-survival and anti-apoptotic effect to alleviate the cellular damages. Thus, the present study proposes a new cellular defensive mechanism during the environmental mutagen and carcinogen attack. [Copyright &y& Elsevier]

Abstract: Human Pol ι is a highly distributed, low-fidelity DNA polymerase lacking intrinsic exonuclease proofreading activity, thus its effects are strictly regulated. We predicted and cloned the promoter region of the human POLI gene. Successively, by transfection of deletion constructs of the POLI promoter, we demonstrated that the regions −848/−408 and −30/+215 contained positive regulatory elements, and the region +215/+335 had proximal promoter activity. Overexpression of Sp1 significantly increased the transcriptional activity of the promoter, and mutation of the Sp1 site reversed Sp1-induced promoter transactivation. Quantitative RT-PCR showed that POLI mRNA expression was up-regulated in human amnion FL cells treated by the carcinogen N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). Reporter gene assays demonstrated that MNNG also significantly increased the transcriptional activity of the predicted promoter (−848/+335) and the proximal promoter (+215/+335). However, the promoter with the Sp1 site mutation had no response to MNNG treatment, suggesting that Sp1 plays an important role in the transcriptional regulation of the POLI gene stimulated by MNNG. Our data suggest that abnormal regulation of Pol ι may be involved in the mutagenesis and carcinogenesis induced by environmental chemicals. [Copyright &y& Elsevier]

Abstract: Two forms of ribonucleotide reductase (RR), consisting of M1 with M2 subunits and M1 with p53R2 subunits, are involved in DNA replication and damage repair, respectively. 3-Aminopyridine-2-carboxaldehyde thiosemicarbazone (3AP), one of the heterocyclic carboxaldehyde thiosemicabazones (HCTs), is a potent RR inhibitor in clinical trial for cancer treatment. In this study, 3AP and its 7 derivatives showed 100–1000-fold higher inhibitory potency on KB nasopharyngeal carcinoma cells than hydroxyurea and were fully active against hydroxyurea- and gemcitabine-resistant KB cells. In vitro RR assays using two recombinant RRs showed that all 8 HCTs decreased the activity of both RRs in a dose-dependent manner and the efficiency was compatible with that on cell proliferation inhibition. Iron has different impact on the behavior of the compounds toward RRs. In the absence of iron, the HCTs showed more selective inhibition for p53R2–M1 than M2–M1, while addition of iron increased their activity but reduced their selectivity for two RRs. Radioligand binding assays showed that [3H]3AP directly bounded to the small subunits. Electron paramagnetic resonance measurements demonstrated that these HCTs generated reactive oxygen species with ferrous iron, which quenched the diiron-tyrosyl radical co-factor of the small subunits and hence the enzyme activity. While the ROS may be a common mediator responsible for the potent activity of the HCTs, the different characteristics of the small subunit proteins are probably associated with the subunit-selectivity of inhibition. Better understanding of the mechanism of action of RR inhibition may improve design of new potent and subunit-selective RR inhibitors for cancer therapy. [Copyright &y& Elsevier]

Abstract: Ribonucleotide reductase (RR) is a highly regulated enzyme in the deoxyribonucleotide synthesis pathway. RR is responsible for the de novo conversion of ribonucleoside diphosphates to deoxyribonucleoside diphosphates, which are essential for DNA synthesis and repair. Besides two subunits, hRRM1 and hRRM2, p53R2 is a newly identified member of RR family that is induced by ultraviolet light in a p53-dependent manner. To understand the molecular interaction of RR subunits, we employed a eukaryotic expression system to express and purify all three subunits. After in vitro reconstitution, the results of [3H]CDP reduction assay showed that both eukaryotic recombinant hRRM2 and p53R2 proteins could interact with hRRM1 to form functional RR holoenzyme. The reconstituted RR activity was time-dependent and the reaction rate reached the plateau phase after 40min incubation. No matter the concentration, RR holoenzyme reconstituted from p53R2 and hRRM1 could only achieve about 40–75% kinetic activity of that from hRRM2 and hRRM1. The synthetic C-terminal heptapeptide competition assays confirmed that hRRM2 and p53R2 share the same binding site on hRRM1, but the binding site on hRRM1 demonstrated higher affinity for hRRM2 than for p53R2. In allosteric regulation assay, the effect of activation or inhibition of hRRM1 with ATP or dATP suggested that these effectors could regulate RR activity independent of different RR small subunits. Taken together, the eukaryotic expression system RR holoenzyme will provide a very useful tool to understand the molecular mechanisms of RR activity and the interactions of its subunits. [Copyright &y& Elsevier]