Anti-MDR Effects of Quercetin and its Nanoemulsion in Multidrug Resistant Human Leukemia Cells

Background: The quercetin has potential against the multidrug resistance (MDR) phenotype, but with low bioavailability. The increase in the bioavailability can be obtained with nanostructures. Objective: To analyze the effects of quercetin and its nanoemulsion on MDR and non-MDR cells. Method: We used: high pressure homogenization for nanoemulsion production; Trypan Blue for cytostatic/cytotoxicity assays; Epifluorescence microscope (with specific probes) for apoptosis and DNA damage; Real Time PCR for gene expression; AutoDock Vina for docking and Flow Cytometry for efflux analysis. The quercetin exerted antiproliferative impact, induced apoptosis, necrosis and DNA damage on cells. Results: Quercetin combined with vincristine showed an effect similar to verapamil (an ABCB1 inhibitor), and the docking showed that bind to ABCB1 in a similar region. The quercetin also was capable of to alter ABCB1 gene expression. The quercetin in nanoemulsion maintained the cytotoxic and cytostatic effects of quercetin which may increase bioavailability. Besides, the unloaded nanoemulsion was able to inhibit per se the efflux activity of ABCB1, demonstrating the pharmacological action of this structure. Conclusion: The quercetin may be considered as a prospective drug to overcome resistance in cancer cells and its nanoemulsion can be an alternative for in vivo application.

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Genotoxicity and Gene Expression in the Rat Lung Tissue following Instillation and Inhalation of Different Variants of Amorphous Silica Nanomaterials (aSiO2 NM)

Nanomaterials ◽

10.3390/nano11061502 ◽

2021 ◽

Vol 11 (6) ◽

pp. 1502

Author(s):

Fátima Brandão ◽

Carla Costa ◽

Maria João Bessa ◽

Elise Dumortier ◽

Florence Debacq-Chainiaux ◽

...

Keyword(s):

Gene Expression ◽

Dna Damage ◽

Amorphous Silica ◽

Oxidative Dna Damage ◽

Intratracheal Instillation ◽

Dna Lesions ◽

Lung Cells ◽

Rat Lung ◽

Inhalation Study

Several reports on amorphous silica nanomaterial (aSiO2 NM) toxicity have been questioning their safety. Herein, we investigated the in vivo pulmonary toxicity of four variants of aSiO2 NM: SiO2_15_Unmod, SiO2_15_Amino, SiO2_7 and SiO2_40. We focused on alterations in lung DNA and protein integrity, and gene expression following single intratracheal instillation in rats. Additionally, a short-term inhalation study (STIS) was carried out for SiO2_7, using TiO2_NM105 as a benchmark NM. In the instillation study, a significant but slight increase in oxidative DNA damage in rats exposed to the highest instilled dose (0.36 mg/rat) of SiO2_15_Amino was observed in the recovery (R) group. Exposure to SiO2_7 or SiO2_40 markedly increased oxidative DNA lesions in rat lung cells of the exposure (E) group at every tested dose. This damage seems to be repaired, since no changes compared to controls were observed in the R groups. In STIS, a significant increase in DNA strand breaks of the lung cells exposed to 0.5 mg/m3 of SiO2_7 or 50 mg/m3 of TiO2_NM105 was observed in both groups. The detected gene expression changes suggest that oxidative stress and/or inflammation pathways are likely implicated in the induction of (oxidative) DNA damage. Overall, all tested aSiO2 NM were not associated with marked in vivo toxicity following instillation or STIS. The genotoxicity findings for SiO2_7 from instillation and STIS are concordant; however, changes in STIS animals were more permanent/difficult to revert.

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MicroRNAs in mouse models of lymphoid malignancies

Journal of Nucleic Acids Investigation ◽

10.4081/jnai.2010.1727 ◽

2010 ◽

Vol 1 (1) ◽

pp. 8 ◽

Cited By ~ 5

Author(s):

Nicola A. O. Zanesi ◽

Yuri Pekarsky ◽

Francesco Trapasso ◽

George Calin ◽

Carlo M. Croce

Keyword(s):

Gene Expression ◽

Mouse Models ◽

Gene Expression Regulation ◽

Human Leukemia ◽

Down Regulation ◽

Over Expression ◽

Lymphoid Malignancies ◽

Knockout Animals ◽

Mouse Modeling

The discovery of microRNAs (miRNAs) has revealed a new layer of gene expression regulation that affects many normal and pathologic biological systems. Among the malignancies affected by the dysregulation of miRNAs there are cancers of lymphoid origin, in which miRNAs are thought to have tumor suppressive or tumor promoting activities, depending on the nature of their specific targets. In the last 4-5 years, the experimental field that provided the deepest insights into the in vivo biology of miRNAs is that of mouse modeling in which transgenic and knockout animals mimic, respectively, over-expression or down-regulation of specific miRNAs involved in human leukemia/lymphoma. This review discusses recent advances in our understanding of lymphoid malignancies based on the natural and engineered mouse models of three different miRNAs, miR-15a/16-1 cluster, miR-155, and miR-17-92 cluster.

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Differential Effects of Chromium(VI) on Constitutive and Inducible Gene Expression in Chick Embryo Liver In Vivo and Correlation With Chromium(VI)-Induced DNA Damage

Molecular Carcinogenesis ◽

10.1002/mc.2940020508 ◽

1989 ◽

Vol 2 (5) ◽

pp. 274-286 ◽

Cited By ~ 56

Author(s):

Joshua W. Hamilton ◽

Karen E. Wetterhahn

Keyword(s):

Gene Expression ◽

Dna Damage ◽

Chick Embryo ◽

Inducible Gene Expression ◽

Differential Effects ◽

Chromium Vi ◽

Embryo Liver ◽

Inducible Gene

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Optimised Genetic Tools Allow the Biosynthesis of Glycocin F and Analogues Designed to Test the Roles of Gcc Cluster Genes in Bacteriocin Production.

Journal of Bacteriology ◽

10.1128/jb.00529-20 ◽

2021 ◽

Author(s):

Brittany J. Drummond ◽

Trevor S. Loo ◽

Mark L. Patchett ◽

Gillian E. Norris

Keyword(s):

Gene Expression ◽

Antimicrobial Agents ◽

Response Regulator ◽

Shuttle Vector ◽

Multidrug Resistant ◽

Target Range ◽

Efficient Production ◽

Bacterial Strains ◽

Natural Product Research

The emergence of multidrug-resistant pathogens has motivated natural product research to inform the development of new antimicrobial agents. Glycocin F (GccF) is a diglycosylated 43-amino acid bacteriocin secreted by Lactobacillus plantarum KW30. It displays a moderate phylogenetic target range that includes vancomycin-resistant strains of Enterococcus species and appears to have a novel bacteriostatic mechanism, rapidly inhibiting growth of the most susceptible bacterial strains at pM concentrations. Experimental verification of the predicted role(s) of gcc cluster genes in GccF biosynthesis has been hampered by the inability to produce soluble recombinant Gcc proteins. Here we report the development of pRV610gcc, an easily modifiable 11.2 kbp plasmid that enables the production of GccF in L. plantarum NC8. Gcc gene expression relies on native promoters in the cloned cluster, and NC8 pRV610gcc produces mature GccF at levels similar to KW30. Key findings are that: the glycosyltransferase glycosylates both serine and cysteine at either position in the sequence, but glycosylation of the loop serine is both sequence and spatially specific; glycosylation of the peptide scaffold is not required for export and subsequent disulfide bond formation; neither of the putative thioredoxin proteins is essential for peptide maturation; removal of the entire putative response regulator GccE decreases GccF production less than removal of the LytTR domain alone. Using this system, we have verified the functions of most of the gcc genes and have advanced our understanding of the roles of GccF structure in its maturation and antibacterial activity. IMPORTANCE The entire 7-gene cluster for the diglycosylated bacteriocin glycocin F (GccF), including the natural promoters responsible for gcc gene expression, has been ligated into the E. coli-LAB shuttle vector pRV610 to produce the easily modifiable 11.2 kbp plasmid pRV610gcc for the efficient production of glycocin F analogues. In contrast to the refactoring approach, chemical synthesis, or chemoenzymatic synthesis, all of which have been successfully used to probe glycocin structure and function, this plasmid can also be used to probe in vivo the evolutionary constraints on glycocin scaffolds and their processing by the maturation pathway machinery, thus increasing understanding of the enzymes involved, the order in which they act and how they are regulated.

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New Shuttle Vectors for Real-Time Gene Expression Analysis in Multidrug-Resistant Acinetobacter Species: In Vitro and In Vivo Responses to Environmental Stressors

Applied and Environmental Microbiology ◽

10.1128/aem.01334-19 ◽

2019 ◽

Vol 85 (18) ◽

Cited By ~ 1

Author(s):

Massimiliano Lucidi ◽

Daniela Visaggio ◽

Elisa Prencipe ◽

Francesco Imperi ◽

Giordano Rampioni ◽

...

Keyword(s):

Gene Expression ◽

Expression Analysis ◽

Gene Expression Analysis ◽

Multidrug Resistant ◽

Antibiotic Selection ◽

Content Type ◽

Shuttle Vectors ◽

Reporter Systems

ABSTRACT The Acinetobacter genus includes species of opportunistic pathogens and harmless saprophytes. The type species, Acinetobacter baumannii, is a nosocomial pathogen renowned for being multidrug resistant (MDR). Despite the clinical relevance of infections caused by MDR A. baumannii and a few other Acinetobacter spp., the regulation of their pathogenicity remains elusive due to the scarcity of adequate genetic tools, including vectors for gene expression analysis. Here, we report the generation and testing of a series of Escherichia coli-Acinetobacter promoter-probe vectors suitable for gene expression analysis in Acinetobacter spp. These vectors, named pLPV1Z, pLPV2Z, and pLPV3Z, carry both gentamicin and zeocin resistance markers and contain lux, lacZ, and green fluorescent protein (GFP) reporter systems downstream of an extended polylinker, respectively. The presence of a toxin-antitoxin gene system and the high copy number allow pLPV plasmids to be stably maintained even without antibiotic selection. The pLPV plasmids can easily be introduced by electroporation into MDR A. baumannii belonging to the major international lineages as well as into species of the Acinetobacter calcoaceticus-A. baumannii complex. The pLPV vectors have successfully been employed to study the regulation of stress-responsive A. baumannii promoters, including the DNA damage-inducible uvrABC promoter, the ethanol-inducible adhP and yahK promoters, and the iron-repressible promoter of the acinetobactin siderophore biosynthesis gene basA. A lux-tagged A. baumannii ATCC 19606T strain, carrying the iron-responsive pLPV1Z::PbasA promoter fusion, allowed in vivo and ex vivo monitoring of the bacterial burden in the Galleria mellonella infection model. IMPORTANCE The short-term adaptive response to environmental cues greatly contributes to the ecological success of bacteria, and profound alterations in bacterial gene expression occur in response to physical, chemical, and nutritional stresses. Bacteria belonging to the Acinetobacter genus are ubiquitous inhabitants of soil and water though some species, such as Acinetobacter baumannii, are pathogenic and cause serious concern due to antibiotic resistance. Understanding A. baumannii pathobiology requires adequate genetic tools for gene expression analysis, and to this end we developed user-friendly shuttle vectors to probe the transcriptional responses to different environmental stresses. Vectors were constructed to overcome the problem of antibiotic selection in multidrug-resistant strains and were equipped with suitable reporter systems to facilitate signal detection. By means of these vectors, the transcriptional response of A. baumannii to DNA damage, ethanol exposure, and iron starvation was investigated both in vitro and in vivo, providing insights into A. baumannii adaptation during stress and infection.

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Interaction between DNA-damage protein GADD34 and a new member of the Hsp40 family of heat shock proteins that is induced by a DNA-damaging reagent

Biochemical Journal ◽

10.1042/bj3520795 ◽

2000 ◽

Vol 352 (3) ◽

pp. 795-800 ◽

Cited By ~ 15

Author(s):

Tadao HASEGAWA ◽

Hengyi XIAO ◽

Fumiyasu HAMAJIMA ◽

Ken-ichi ISOBE

Keyword(s):

Dna Damage ◽

Heat Shock ◽

Cultured Cells ◽

Similar Region ◽

C Terminus ◽

Mouse Tissues ◽

Time Courses ◽

Shock Proteins ◽

Two Hybrid

GADD34 is one of a subset of proteins induced after DNA damage or cell growth arrest. To examine the function of GADD34, we used the yeast two-hybrid system to clone the protein that interacts with murine GADD34. As bait we used the product of the partial GADD34 cDNA, including the regions rich in proline, glutamic acid, serine and threonine (PEST) and γ134.5 regions. A cDNA clone, named GAHSP40, which is a mouse DnaJ family protein with a high similarity to human HLJ1 was cloned. The interaction between GADD34 and GAHSP40 in cultured cells was confirmed by a co-immunoprecipitation experiment and in NIH 3T3 cells by two-hybrid analysis in vivo. For binding of the two proteins, the γ134.5-similar region of GADD34 was necessary; however, the PEST region was also involved and the C-terminus of GAHSP40, but not the J-domain, was important. GAHSP40 was detected in all mouse tissues examined, but a different transcript was found in the testis. Both GADD34 mRNA and GAHSP40 mRNA were significantly elevated by treatment with methyl methanesulphonate, although the time courses were different. In addition, both GAHSP40 and GADD34 mRNA were induced by heat shock.

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Col-6, a Compound Found in Edible Plants, Induces Apoptosis of Human Leukemia Cells and Down-Regulate Bcl-2 Expression.

Blood ◽

10.1182/blood.v104.11.4380.4380 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4380-4380

Author(s):

Xudong Ma ◽

A. Beklemisheva ◽

Delong Liu ◽

J.W. Chiao

Keyword(s):

Mononuclear Cells ◽

Leukemic Cell ◽

G1 Arrest ◽

Human Leukemia ◽

Edible Plants ◽

Proliferating Cells ◽

Normal Peripheral Blood ◽

High Concentrations ◽

Induced Apoptosis

Abstract Col-6 is a compound found in certain edible plants. Col-6 has been synthesized, and structurely-characterized. This study investigated its effects on the growth of human promyelocytic leukemic cell line, HL-60. Exposure of HL-60 cells to Col-6 at high concentrations (≥40 μM) resulted in cytolysis in 90% cells within 72 hours. At lower concentrations (<20 μM) the cell density and viability were reduced as compared to untreated cells. A 30% reduction in cell growth rate was seen at 10 μM within 48 hours. A reduction of the proliferating cells in S and G2M phases along with an increase of G1 were detected, indicating a G1 arrest. Multiple assays were used to characterize apoptosis, including DNA strand breaks by TUNEL assay, and the presence of sub-G1 peak by flow cytometry. Col-6 induced apoptosis of HL-60 cells regularly at a concentration as low as 0.1 μM. The degree of apoptosis was proportional to the concentrations and the length of exposure. Mononuclear cells isolated from normal peripheral blood (PBMC) and bone marrow (BMMC) were similarly exposed to Col-6. At a concentration as high as 40 μM, Col-6 did not cause apoptosis of PBMC nor BMMC, suggesting that Col-6 does not have the apoptotic effects on normal non-dividing cells. Apoptosis was further correlated with the increase of caspase 9, and the cleavage of PARP (substrate of caspases), as defined by Western blot analyses. The expression of bcl-2 was markedly decreased in the treated cells. Conclusion: Col-6, a compound found in edible plants, induced apoptosis of HL-60 cells and down-regulation of bcl-2. Col-6 may be a potential novel anti-leukemia agent. Further in vivo study is indicated.

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Loss Of IKZF1 Function Mediates Resistance Towards Glucocorticoid-Induced Apoptosis

Blood ◽

10.1182/blood.v122.21.3865.3865 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3865-3865

Author(s):

Jorn Havinga ◽

Laurensia Yuniati ◽

Marc Demkes ◽

Dorette van Ingen Schenau ◽

Roland P. Kuiper ◽

...

Keyword(s):

Gene Expression ◽

Prognostic Factor ◽

Transcriptional Activation ◽

Lymphoblastic Leukemia ◽

Luciferase Reporter ◽

Human Leukemia ◽

Wild Type ◽

Gene Deletions ◽

Expression Levels ◽

Induced Apoptosis

Abstract Background Glucocorticoids (GCs) such as prednisolone and dexamethasone are critical components of multi-agent chemotherapy regimens used in the treatment of acute lymphoblastic leukemia (ALL). Children with ALL are stratified into risk groups based on diagnostic features (i.e. age and cytogenetics) and therapy response. It has been established that the initial response to prednisolone is a major prognostic factor. Moreover, at relapse, de novo or acquired resistance to GCs is common and represents an important determinant in treatment failure. Recent studies performed by us and others have identified IKZF1 gene deletions and mutations as an independent prognostic factor that predicts prognosis and treatment outcome of children with B cell precursor ALL (BCP-ALL). These monoallelic IKZF1 gene deletions either affect the whole gene or may result in expression of dominant-negative IKZF1 isoforms due to intragenic deletions. However, it has not been established whether loss of IKZF1 function directly impacts the response to glucocorticoids. Results We examined whether haplodeficiency for Ikzf1 gene expression in mouse lymphocytes affects glucocorticoid-induced apoptosis. Splenocytes from Ikzf1+/- knockout mice were activated with lipopolysaccharide (LPS) and treated with increasing concentrations of either prednisolone or dexamethasone for 48 hours. B-lymphocytes haplodeficient for IKZF1 showed a significantly enhanced survival after treatment with GCs compared to wild type cells, as measured in an MTS assay and by AnnexinV staining. In case of prednisolone, the inhibitory concentration (IC50) was about ∼200-fold higher in the Ikzf1+/- splenocytes as compared to the wild-type cells. Gene expression analysis revealed that Ikzf1+/- splenocytes displayed lower overall expression levels as well as diminished transcriptional activation of several glucocorticoid receptor (GR)-induced target genes (i.e. Sgk1, Irs2, Zfp36L2). Furthermore, in luciferase reporter assays we established that IKZF1 overexpression enhances GR-mediated transcriptional activation in response to prednisolone. Finally, lentivirus-mediated IKZF1-shRNA expression in Nalm6 cell line, which reduces endogenous IKZF1 protein levels to around 50%, inhibits prednisolone and dexamethasone-induced apoptosis, demonstrating that also in human leukemia cells reduced IKZF1 expression levels protect against GC-induced cell death. In conclusion, our data provide evidence that loss of IKZF1 function mediates resistance to glucocorticoid-induced apoptosis, which may contribute to the poor outcome of IKZF1-deleted BCP-ALL. Disclosures: No relevant conflicts of interest to declare.

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Transcriptional corepressor MTG16 regulates small intestinal crypt proliferation and crypt regeneration after radiation-induced injury

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00253.2014 ◽

2015 ◽

Vol 308 (6) ◽

pp. G562-G571 ◽

Cited By ~ 14

Author(s):

Shenika V. Poindexter ◽

Vishruth K. Reddy ◽

Mukul K. Mittal ◽

Amanda M. Williams ◽

M. Kay Washington ◽

...

Keyword(s):

Dna Damage ◽

Stem Cell ◽

Epithelial Cells ◽

Cellular Response ◽

Cell Function ◽

Enterocyte Proliferation ◽

Induced Apoptosis ◽

Chemical Colitis ◽

Transcriptional Corepressors

Myeloid translocation genes (MTGs) are transcriptional corepressors implicated in development, malignancy, differentiation, and stem cell function. While MTG16 loss renders mice sensitive to chemical colitis, the role of MTG16 in the small intestine is unknown. Histological examination revealed that Mtg16 −/− mice have increased enterocyte proliferation and goblet cell deficiency. After exposure to radiation, Mtg16 −/− mice exhibited increased crypt viability and decreased apoptosis compared with wild-type (WT) mice. Flow cytometric and immunofluorescence analysis of intestinal epithelial cells for phospho-histone H2A.X also indicated decreased DNA damage and apoptosis in Mtg16 −/− intestines. To determine if Mtg16 deletion affected epithelial cells in a cell-autonomous fashion, intestinal crypts were isolated from Mtg16 −/− mice. Mtg16 −/− and WT intestinal crypts showed similar enterosphere forming efficiencies when cultured in the presence of EGF, Noggin, and R-spondin. However, when Mtg16 −/− crypts were cultured in the presence of Wnt3a, they demonstrated higher enterosphere forming efficiencies and delayed progression to mature enteroids. Mtg16 −/− intestinal crypts isolated from irradiated mice exhibited increased survival compared with WT intestinal crypts. Interestingly, Mtg16 expression was reduced in a stem cell-enriched population at the time of crypt regeneration. This is consistent with MTG16 negatively regulating regeneration in vivo. Taken together, our data demonstrate that MTG16 loss promotes radioresistance and impacts intestinal stem cell function, possibly due to shifting cellular response away from DNA damage-induced apoptosis and towards DNA repair after injury.

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