Identification and Characterisation of a Novel Antioxidant Activity for the BCAT1 Cxxc Motif: Implications for Myeloid Leukaemia Development

Abstract Recently the human cytosolic branched-chain amino transferase (BCAT1) has been implicated in the development of myeloid leukaemia. Previously studies identified a redox active CXXC motif for BCAT1 analogous to the mitochondrial isoform (BCAT2). Oxidation of this CXXC motif can regulate aminotransferase activity in both isoforms. Interestingly the reduction mid-point potential (Em) of the BCAT1 CXXC motif is around 80mV lower compared with BCAT2. This suggests that the BCAT1 CXXC motif is more reducing in nature i.e. has antioxidant capacity. Further evidence to support this notion comes from the observation that BCAT1 is significantly less sensitive to inactivation mediated by hydrogen peroxide (H2O2). This is an important observation, since H2O2 is a reactive oxygen species (ROS) implicated in myeloid leukaemia development. Here we provide the first evidence to show that the BCAT1 CXXC can metabolise H2O2. By using CXXC motif mutant constructs, we also evaluate the role of the BCAT1 CXXC motif in myeloid leukaemia cells. To investigate the antioxidant capacity of the BCAT1 CXXC motif, cDNA (accession NM_001178094.1) was cloned into a pET28a vector for overexpression and purification in E.coli BL21(DE3) cells. Site directed mutagenesis of the CXXC motif subsequently followed creating three constructs: 1) BCAT1-WT (unmutated), 2) BCAT1-CXXS (C-terminal Cys → Ser mutant) and 3) BCAT1-SXXS (N/C-terminal Cys → Ser double mutant). Mutation of the BCAT1 CXXC motif was confirmed at the protein level by 5,5'-ditho-bis-(2-nitrobenzoic acid) titration. WT and CXXC motif mutant BCAT1 protein was added to 5mM H2O2 & monitored at λ240nm for the disappearance of H2O2. This was compared with 1U of catalase as a positive control. Our data show that 15μg of purified BCAT1-WT could metabolise H2O2 at a rate of 0.14±0.02 μmol/min. The BCAT1 CXXC motif mutants lacked capacity to do this. To verify our findings, BCAT1-WT treated with N-ethylmaleimide also lost the capacity to metabolise H2O2. This confirms that the antioxidant activity observed is Cys mediated. This novel finding for the BCAT1 CXXC motif may therefore be important in myeloid leukaemia development. To evaluate the BCAT1 CXXC motif in myeloid leukaemia cells, WT and CXXS mutant BCAT1 was subcloned into a pLENTI-C-Myc-DDK lentiviral vector prior to transduction of U937 cells. Empty vector (EV) control transgenic U937 cells were also generated. Stable expression of BCAT1 was achieved and confirmed by western-blot and qPCR; BCAT1-WT (6.5±1.7 fold increase) & BCAT1-CXXS (5.1±2.6 fold increase). The BCAT1 and EV transgenic U937 cells were subjected to H2O2 mediated oxidative stress and monitored for cell viability after 24h. A significant difference in the LD50 between BCAT1-WT, BCAT1-CXXS and EV control cells was observed; 620±28mM, 306±37mM, 251±26mM respectively (p<0.05). This finding suggests that the BCAT1 CXXC provides protection against H2O2 mediated cell death. To corroborate these findings, the same cells treated with rotenone observed a similar pattern in LD50; 1.05mM, 0.06mM, 0.12mM respectively. ROS are implicated in many cellular processes, including differentiation. Thus, we next asked whether the BCAT1 CXXC motif could suppress U937 cell differentiation mediated by phorbol 12-myristate 13-acetate (PMA). This compound was selected since ROS feature in PMA mediated differentiation. U937 cells were monitored for the expression of CD11b and CD36 following 48h PMA incubation. Our data show a significant reduction in the frequency of CD11b+/CD36+ U937 cells for BCAT1-WT (17.5±6.9%) compared with EV controls (54.0±10.5%, p<0.05). BCAT1-CXXS also showed a reduction (30.6±4.9%), but not to the same extent as BCAT1-WT. This finding was mirrored by the expression level of CD11b, where the MFI was significantly less for BCAT1-WT (88.0±16.3) compared with EV control (251.5±54.7, p<0.001) and BCAT1-CXXS (152.5±6.3, p<0.05). Analysis of intracellular ROS using 2′,7′-Dichlorodihydrofluorescein diacetate revealed a reduction in cellular ROS for BCAT1-WT cells (5929±13.8 MFI), compared with EV controls (8527±196 MFI) and BCAT1-CXXS (7879±18.4 MFI). This shows that the BCAT1 CXXC motif may control cellular ROS levels in myeloid leukaemia cells. In summary, this study identifies a novel antioxidant role for the BCAT1 CXXC which confers protection against ROS mediated cell death and differentiation of myeloid leukaemia cells. Disclosures No relevant conflicts of interest to declare.

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ROCK1 Functions as a Suppressor of Inflammatory Cell Migration by Regulating PTEN Phosphorylation and Stability.

Blood ◽

10.1182/blood.v114.22.240.240 ◽

2009 ◽

Vol 114 (22) ◽

pp. 240-240

Author(s):

Sasidhar Vemula ◽

Jianjian Shi ◽

Philip Hanneman ◽

Lei Wei ◽

Reuben Kapur

Keyword(s):

Bone Marrow ◽

Acute Inflammation ◽

Conflicts Of Interest ◽

Flow Cytometric Analysis ◽

Fold Increase ◽

Phagocytic Index ◽

In Vivo Model ◽

Significant Difference ◽

Rho Kinases

Abstract Abstract 240 Neutrophils and macrophages are major cellular components of the innate immune response and are recruited rapidly in large numbers to sites of infection. The small family of Rho GTPases and its downstream effectors, Rho kinases (Rho-associated, coiled-coil containing protein kinase) have been implicated in regulating various cellular functions including actin cytoskeleton organization, cell adhesion, and cell motility in non-hematopoietic cells. Rho kinases (ROCK1 and ROCK2) belong to a family of serine/threonine kinases whose role in inflammation is not known. Here we show that deficiency of ROCK1 but not ROCK2 results in increased recruitment of macrophages (3.2 fold, n=8, *p<0.01) and neutrophils (3.4 fold, n=5 *p<0.05) compared to WT controls in an in vivo model of aseptic peritonitis. In vitro, deficiency of ROCK1 in bone marrow derived macrophages shows a significant increase in haptotactic transwell migration in response to M-CSF as well as MCP-1 on fibronectin as well as an increase in migration towards the wounded area in a wound healing assay compared to controls (∼3 fold, n=3, *p<0.005). Consistently, deficiency of ROCK1 in bone marrow derived neutrophils also shows a ∼2.63 fold increase in migration in response to fMLP compared to WT bone marrow derived neutrophils (BMNs) in a chemotactic migration assay. ROCK1 deficient macrophages also demonstrate a ∼2.5 fold increase in adhesion on fibronectin (n=3, *p<0.002). The enhanced migration and adhesion in ROCK1−/− macrophages was observed in spite of comparable expression of F4/80 (WT; 85.63% vs. ROCK1−/−; 88.68%, n=4), α4β1 and α5β1 integrins (WT; 67.49% & 88.2% vs. ROCK1−/−; 71.82 % & 87.09%, n=4), while no significant difference in the phagocytosis of sheep red blood cells was observed between WT and ROCK1−/− macrophages (Phagocytic index: WT; 98% vs. ROCK1−/− 97%, n=3, p>.05). Close examination of the cytoskeleton of ROCK1 deficient macrophages using confocal microscopy revealed more F-actin content on the entire cell surface compared to wildtype controls. Consistently, flow cytometric analysis using Alexa 488-phalloidin staining revealed abundance of F-actin in ROCK1−/− macrophages compared to WT controls (WT; 46.19% vs. ROCK1−/−; 65.23%, n=3, *p<0.05). Furthermore, immunofluorescence imaging of podosomes carried out using anti-vinculin antibody revealed more pronounced and increased podosomes in ROCK1 deficient macrophages compared to WT controls (n=3, *p<0.05). Biochemical analysis of ROCK1−/− macrophages revealed that the enhanced recruitment of ROCK1 deficient macrophages and neutrophils was apparent in spite of normal expression of ROCK2 in ROCK1−/− cells and a 60% reduction in overall ROCK activity. Interestingly, although both ROCK1 and ROCK2 co-immunoprecipitate with PTEN in response to cytokine induced stimulation, only ROCK1 appeared to be essential for PTEN phosphorylation, activation and stability. In the absence of ROCK1, PTEN phosphorylation, its activity and stability were significantly impaired in spite of the presence of ROCK2 (n=3, *p<0.05). Consequently, an increase in the activation of downstream targets of PTEN including AKT, GSK-3β and cyclinD1 was observed in ROCK1 deficient macrophages relative to controls (n=3). Taken together, these studies reveal a biochemical pathway involving ROCK1 and PTEN which is involved in the recruitment of macrophages and neutrophils during acute inflammation. Thus, ROCK1 likely functions as a physiologic regulator of PTEN whose function is to repress excessive recruitment of macrophages and neutrophils during acute inflammation. Disclosures: No relevant conflicts of interest to declare.

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Increased Use of Allogeneic Transplant in CR2 Improves the Outcome for Children with Acute Myeloid Leukaemia

Blood ◽

10.1182/blood.v126.23.2521.2521 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2521-2521

Author(s):

Aditi Vedi ◽

Richard Mitchell ◽

Cecelia Oswald ◽

Glenn M Marshall ◽

Toby Trahair ◽

...

Keyword(s):

Acute Myeloid Leukaemia ◽

Supportive Care ◽

Myeloid Leukaemia ◽

De Novo ◽

Conflicts Of Interest ◽

Induction Treatment ◽

Allogeneic Transplant ◽

Significant Difference ◽

Related Mortality ◽

Acute Myeloid

Abstract Improvements in Outcome for Paediatric de novo Acute Myeloid Leukaemia Aditi Vedi1,2, Richard Mitchell1, Cecelia Oswald1, Glenn Marshall1,2, Toby Trahair1, David S Ziegler1,2 1Kids Cancer Centre, Sydney ChildrenÕs Hospital, Randwick, NSW, Australia, 2 School of Women and Children's Health, University of New South Wales, Randwick, NSW, Australia ABSTRACT The treatment for paediatric acute myeloid leukaemia (AML) has not changed significantly over the past 3 decades, yet outcomes have improved with cure rates increasing from 30% to over 50% of all newly diagnosed children over this period. This improvement in survival has been attributed to both treatment intensification and improved supportive care over the decades, although the precise impact of each remains unknown. Our group has retrospectively analysed a unique cohort of patients with de novo AML diagnosed in childhood (n=276), all treated with the same chemotherapy protocol over a 25-year period from 1986-2012. The contemporary cohort (2000-12), compared to historical cohorts (1986-99) had significantly improved overall survival (OS, 75% vs. 50%, p = 0.01), lower disease related mortality (38% vs. 19%, p = 0.02) and were significantly more likely to receive allogeneic transplant after relapse (SCT, 73% vs. 12%, p <0.0001). Allogeneic transplant post relapse was associated with a significantly improved survival across the entire cohort (OS 50% for allogeneic SCT vs. 12% for autologous or none, p<0.0001). There was no significant difference between the contemporary and historical cohorts in treatment related mortality (13% vs. 7%, p = 0.42) or relapse rates after induction (50% in older cohort vs. 40% in recent era, p=0.25), suggesting consistency of induction treatment efficacy and toxicity across the two periods. This data suggests improved survival in paediatric AML in the modern era has predominantly resulted from increased use of allogeneic SCT after relapse rather than from improved supportive care and is independent of chemotherapy intensification. Disclosures No relevant conflicts of interest to declare.

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Ethanolic extract of mango seed used in the feeding of broilers: effects on phenolic compounds, antioxidant activity, and meat quality

Canadian Journal of Animal Science ◽

10.1139/cjas-2018-0120 ◽

2020 ◽

Vol 100 (2) ◽

pp. 299-307

Author(s):

Nadja Naiara Pereira Farias ◽

Ednardo Rodrigues Freitas ◽

Herbenson Marques Gomes ◽

Davyd Herik Souza ◽

Edibergue Oliveira dos Santos ◽

...

Keyword(s):

Antioxidant Activity ◽

Phenolic Compounds ◽

Antioxidant Capacity ◽

Meat Quality ◽

Control Diet ◽

Ethanolic Extract ◽

Butylated Hydroxytoluene ◽

Significant Difference ◽

Mango Seed ◽

Different Levels

The objective of this study was to evaluate the effect of including different levels of ethanolic extract of mango seed (EEMS) in broiler chicken rations on their phenolic compound levels, antioxidant activity, and meat quality. Initially, 756 one-day-old male chicks of the Ross 308 line were distributed in a completely randomized experimental design with seven treatments and six replicates of 18 birds. The treatments consisted of (i) a ration without the addition of antioxidants, (ii) a ration with the addition of 200 ppm of the antioxidant butylated hydroxytoluene (BHT), and (iii) a ration with the addition of 200, 400, 600, 800, or 1000 ppm of EEMS. According to the results, the values of phenolic compounds, the lipid stability of meat (measured by the value of thiobarbituric acid reactive substances), shear force, loss of cooking water, color, and pH of the meat differed significantly between the treatments. For the antioxidant activity of the meat, there was no significant difference between the treatments by the 2,2-diphenyl-1-picrylhydrazyl method; however, by the 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) method, the breast meat of broilers fed diets containing EEMS from 600 ppm and up presented higher antioxidant capacity when compared with the meat of the birds fed the control diet. In the ABTS method, there was no significant difference between the use of synthetic antioxidant BHT and the addition of EEMS at different levels. In conclusion, the addition of EEMS does not affect meat quality parameters of broilers, but when a dose of 600 ppm or more is added, the antioxidant capacity of meat measured by the ABTS method increases.

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Elucidation of the Roles of Individual Asparagine-Linked Glycans Outside of the B Domain on Factor VIII Secretion

Blood ◽

10.1182/blood.v118.21.2238.2238 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2238-2238 ◽

Cited By ~ 1

Author(s):

Sundar Rajan Selvaraj ◽

Hongzhi Miao ◽

Steven Pipe

Keyword(s):

Dendritic Cells ◽

Factor Viii ◽

Conflicts Of Interest ◽

Site Directed Mutagenesis ◽

Significant Difference ◽

C1 Domain ◽

Protein Backbones ◽

And Function

Abstract Abstract 2238 Post-translational modifications play vital roles in the secretion, function, intermolecular interactions and degradation of most secreted and transmembrane proteins. Factor VIII (FVIII) is a heavily glycosylated protein with up to 25 asparagine (Asn)-linked glycans, the bulk of which are present within its B domain. However, deletion of the B domain is not deleterious to FVIII expression and function. In addition, FVIII has several potential Asn-linked glycosylation sequons in its other domains, of which four have been experimentally deduced to be glycosylated: Asn41 and Asn239 in the A1 domain, Asn1810 in the A3 domain and Asn2118 in the C1 domain. Of these, Asn239 and Asn2118 have been determined to comprise complex oligomannose structures. Such complex oligomannose structures have been proposed to play a role in mediating interaction with immunomodulatory cells (i.e. dendritic cells). The present study was aimed at delineating the role(s) of these four Asn-linked glycans in the expression of FVIII in vitro and in vivo and to identify possible bioengineering targets to influence FVIII expression, clearance and processing by immunomodulatory cells. Individual Asn residues were mutated to glutamine (Gln) to create single and multiple glycosylation mutants in both full length (FVIII-WT) and B domain-deleted (BDD)–FVIII, by site-directed mutagenesis. A variant of BDD-FVIII completely devoid of Asn-linked glycans, designated as Degly-BDD-FVIII, was also generated. Transient transfections of the mutants were carried out in COS-1 and CHO cells and their secretion and function were analyzed and compared to that of the respective native FVIII proteins. Antigen and activity assays revealed that the secretion and function of Asn41Gln and Asn1810Gln mutants were only modestly affected (85–90% of WT) but a more significant reduction was observed in the case of Asn239Gln mutant (35–50% of WT). Interestingly, there was no significant difference in secretion or function for Asn2118Gln in either FVIII-WT or BDD-FVIII protein backbones. The double mutants, Asn41/239Gln and Asn239/2118Gln behaved similarly to that of Asn239Gln mutant (30–45% of WT). The triple mutants, Asn41/239/2118Gln and Asn239/1810/2118Gln showed a further decline in secretion (∼30-40% of WT) while Degly-BDD-FVIII demonstrated secretion of only about 15–20% of BDD-FVIII. The FVIII specific activity of each of these glycosylation mutants was similar to the native FVIII proteins. An ELISA-based Von Willebrand Factor (VWF) binding assay revealed no significant differences between immunoaffinity-purified FVIII-WT and Asn2118Gln mutant in their ability to bind VWF. Findings from in vivo expression (via hydrodynamic tail vein injection of plasmid DNA) of these glycosylation mutants in a F8−/− (exon 16 knock-out) hemophilia A mouse model were similar to the in vitro results in the cell lines. Plasma FVIII activity levels were measured 24 hrs post-injection via orbital bleed. While Asn2118Gln (5.2 – 6 U/mL) did not exhibit any difference from BDD-FVIII (4.8 – 5.9 U/ml), Asn239Gln (1.9 – 2.4 U/ml) was expressed at less than 50% of BDD-FVIII levels. The expression of Degly-BDD-FVIII (0.4 – 0.7 U/ml) was further reduced to ∼10% of BDD-FVIII levels. Taken together, these results indicate that of the four Asn-linked glycans, Asn239 was the most crucial for proper secretion of FVIII whereas, Asn2118 did not contribute to the efficiency of FVIII expression. The oligosaccharide structure on Asn239 is positioned at the A1-A2 interface and likely contributes to proper protein folding. However, the sugar moieties on Asn2118 have been shown to be positioned at the A3-C1 domain interface and postulated to participate in packing and stabilization (Shen et al, 2008). This would have suggested that disruption of this residue within the C1 domain might have a deleterious effect on protein secretion or function. Our results with Asn2118Gln in both FVIII-WT and BDD-FVIII protein backbones suggest that this Asn-linked glycosylation can be eliminated without any impact on FVIII expression or function including no impact on FVIII-VWF interaction. This Asn-linked glycan, therefore, could be targeted in bioengineering strategies to determine if eliminating this particular oligomannose structure might impact mannose-receptor mediated uptake of FVIII by dendritic cells. Disclosures: No relevant conflicts of interest to declare.

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Antioxidant Activity of Edible Fungi (Truffles and Mushrooms): Losses during Industrial Processing

Journal of Food Protection ◽

10.4315/0362-028x-65.10.1614 ◽

2002 ◽

Vol 65 (10) ◽

pp. 1614-1622 ◽

Cited By ~ 81

Author(s):

M. ANTONIA MURCIA ◽

MAGDALENA MARTÍNEZ-TOMÉ ◽

ANTONIA M. JIMÉNEZ ◽

ANA M. VERA ◽

MARIO HONRUBIA ◽

...

Keyword(s):

Antioxidant Activity ◽

Antioxidant Capacity ◽

Antioxidant Properties ◽

Trolox Equivalent Antioxidant Capacity ◽

Radical Scavenger ◽

Edible Fungi ◽

Industrial Processing ◽

Radical Scavenger Activity ◽

Scavenger Activity ◽

Significant Difference

The antioxidant properties of two raw truffles (Terfezia claveryi Chatin and Picoa juniperi Vittadini) and five raw mushrooms (Lepista nuda, Lentinus edodes, Agrocybe cylindracea, Cantharellus lutescens, and Hydnum repandum) were tested by subjecting these truffles and mushrooms to different industrial processes (freezing and canning) and comparing them with common food antioxidants (α-tocopherol [E-307], BHA [E-320], BHT [E-321], and propyl gallate [E-310]) with regard to their ability to inhibit lipid oxidation. All of the truffles and mushrooms analyzed exhibited higher percentages of oxidation inhibition than did the food antioxidants according to assays based on lipid peroxidation (LOO•), deoxyribose (OH•), and peroxidase (H2O2). Frozen samples exhibited a small reduction in free radical scavenger activity, but the results did not show a significant difference (P < 0.05) with respect to the raw samples, while canned truffles and mushrooms lost some antioxidant activity as a consequence of industrial processing. All of the raw and frozen truffles and mushrooms except frozen Cantharellus improved the stability of oil against oxidation (100°C Rancimat), while canned samples accelerated oil degradation. Antioxidant activity during 30 days of storage was measured by the linoleic acid assay, and all of the samples except canned Terfezia, Picoa, and Hydnum showed high or medium antioxidant activity. The Trolox equivalent antioxidant capacity assay was used to provide a ranking order of antioxidant activity as measured against that of Trolox (a standard solution used to evaluate equivalent antioxidant capacity). The order of raw samples with regard to antioxidant capacity was as follows (in decreasing order): Cantharellus, Agrocybe, Lentinus, Terfezia, Picoa, Lepista, and Hydnum. Losses of antioxidant activity were detected in the processed samples of these truffles and mushrooms.

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Comparison of phenolic content and antioxidant capacity of red and yellow onions.

Czech Journal of Food Sciences ◽

10.17221/566/2012-cjfs ◽

2013 ◽

Vol 31 (No. 5) ◽

pp. 501-508 ◽

Cited By ~ 28

Author(s):

A. Cheng ◽

X. Chen ◽

Q. Jin ◽

W. Wang ◽

J. Shi ◽

...

Keyword(s):

Antioxidant Activity ◽

Antioxidant Capacity ◽

Phenolic Content ◽

Colorimetric Method ◽

Acid System ◽

Dpph Assay ◽

Total Polyphenol ◽

Significant Difference ◽

Red Variety ◽

Yellow Onion

The total polyphenol (TP), flavonoid, proanthocyanidin (PAC) content, and antioxidant capacity of both onion varieties (red and yellow) were compared. The content of TP, flavonoids, and PAC was determined by Folin-Ciocalteu colorimetric method, AlCl<sub>3</sub>, and by DMAC colorimetric method, respectively. The results showed that the contents of TP and flavonoids decreased from the outer to the inner layers in both onions, but there was no significant difference in PAC content. The outer layers had the highest antioxidant activity of extracts followed by a continuous decrease towards the inner layers in both varieties. The contents of phenolic acids and flavonoids were quantified by HPLC. Gallic acid, ferulic acid, and quercetin, as the main compounds in polyphenols, were detected in each layer of both onions. The red variety showed better antioxidant activity than yellow onion according to the linoleic acid system and DPPH assay. The higher contents of TP and flavonoids were associated with higher antioxidant activity.

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Induction of Senescence by Doxorubicin Is Associated with Upregulated Mir-375 and Induction of Autophagy In CML Cell Line K562

Blood ◽

10.1182/blood.v116.21.1843.1843 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1843-1843

Author(s):

Ming-Yu Yang ◽

Wen-Chi Yang ◽

Pai-Mei Lin ◽

Jui-Feng Hsu ◽

Hui-Hua Hsiao ◽

...

Keyword(s):

Cell Death ◽

Cell Line ◽

Target Genes ◽

Conflicts Of Interest ◽

Expression Patterns ◽

K562 Cells ◽

Fold Increase ◽

Alternative Mode ◽

Telomere Attrition

Abstract Abstract 1843 Senescence is a specialized form of growth arrest that it is generally irreversible and can be induced by telomere attrition, oxidative stress, oncogene expression and DNA damage signaling. Senescent cells display a typical upregulated senescence-associated (SA)-b-galactosidase activity and novel changes in chromatin architecture, the formation of SA heterochromatic foci (SAHF). Changes in gene expression, such as upregulated p16, p53, and p21 expression and silencing of E2F target genes, have been characterized to promote the establishment of senescence. Besides, by the actions of HP1g, HMGA, and DNMT proteins to produce a repressive chromatin environment, the transcription of proliferation-associated genes can be suppressed. Therefore, senescence has been suggested to functions as a natural brake to tumor development. In this study, we first sought to establish an in vitro senescence model using doxorubicin (DOX) and paclitaxel to treat CML cell line K562. We found that 50 nM DOX induced senescence, but did not induce apoptosis. In contrast, 10 nM and 100 nM paclitaxel induced apoptosis but did not induce senescence, and lower doses of paclitaxel (1 nM and 5 nM) had no effect on the cells. p53 and p16-pRb are the two major senescence pathways. Since p53 and p16 are homozygously deleted in the K562 cells, the DOX-induced senescence in K562 cells is supposed to be established by a pathway independent of p53 and p16-pRb pathways. Indeed, the expression of the typical SA-premalignant cell markers (CDC6, Ki67, p19, p38, PU1, DNMT1, HMGA1, HP1g) did not change in the DOX-induced senescent K562 cells although the typical SA-b-galactosidase staining and SAHF were apparent. MicroRNA profiling revealed that miR-375 was upregulated in DOX-induced senescent K562 cells. Treatment with miR-375 inhibitor could rescue the proliferation ability suppressed by DOX (p < 0.05). The identification of miR-375 targets should help us to elucidate the substitution pathway that is responsible for the DOX-induced senescence in the absence of both p16 and p53 genes. With the observation that DOX treatment induced cells entering senescence but eventually lead to cell death, we also investigated if the alternative mode of cell death, autophagy, was involved. By examining the expression patterns of the 26 human autophagy-related (ATG) genes, a 12-fold increase of ATG9B at day 4 and a 20-fold increase of ATG18 at day 2 after DOX treatment were noted. Both ATG9 and ATG18 are crucial for the formation of autophagosome. Hence, in addition to the upregulation of miR-375, our results also demonstrated that senescence induced by DOX in K562 cells is associated with the initiation of autophagy. Updated results on the identification of miR-375 targets and the regulation of senescence and autophagy pathway will be presented at the meeting. Disclosures: No relevant conflicts of interest to declare.

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The Effect of Bortezomib (B) Alone or in Combination with Other Agents for Stem Cell Mobilization in Mice

Blood ◽

10.1182/blood.v120.21.583.583 ◽

2012 ◽

Vol 120 (21) ◽

pp. 583-583

Author(s):

Armin Ghobadi ◽

Matthew Holt ◽

Julie Ritchey ◽

John F. DiPersio

Keyword(s):

Stem Cell ◽

Conflicts Of Interest ◽

Group Versus ◽

Fold Increase ◽

Stem Cell Mobilization ◽

Control Group ◽

Hematopoietic Stem ◽

Significant Difference ◽

Cell Mobilization ◽

Phosphate Buffered Saline

Abstract Abstract 583 Introduction: Granulocyte colony-stimulating factor (G-CSF) is the most commonly used drug for stem cell mobilization. Unfortunately, 5–30% of patients fail to collect sufficient hematopoietic stem/progenitor cells (HSPCs) necessary for transplant. New strategies are needed to increase HSPCs collection in these patients. Apart from cytotoxic effect, bortezomib decreases expression of cell adhesion molecules including VCAM-1. Therefore, we hypothesize that bortezomib can mobilize HSPCs. Method: C57BL/6 (B6) mice were injected with intravenous (IV) bortezomib (0.8 mg/kg) or Phosphate buffered saline (PBS). Blood were harvested at baseline and 12, 15, 18, 21, and 24 hours (h) after injections and plated on MethoCult media (StemCell Technologies). For evaluation of mechanism and source of mobilized HSPCs, bortezomib versus PBS experiments were performed in splenectomized B6 mice and VLA4 knockout (VLA4KO) mice in addition to B6 mice. Experiments involving combination of bortezomib with G-CSF, AMD3100, and Cytoxan were also conducted in B6 mice. In bortezomib-G-CSF group (BG), bortezomib was given on day1 and G-CSF (250 μg/kg subcutaneously) was given on days 2, 3, 4, and 5. In G-CSF-bortezomib group (GB), G-CSF was given on days 2, 3, 4, and 5 and bortezomib was given on day 5. G-CSF (G) control group received G-CSF on days 2, 3, 4, and 5. In bortezomib-AMD3100 group (BA), bortezomib was given at baseline and AMD3100 (5 mg/kg subcutaneously) was given 15 h after bortezomib. In PBS-AMD3100 control group (PA), bortezomib at baseline in BA group was substituted with PBS. In cyclophosphamide-G-CSF group (CG), cyclophosphamide (200 mg/kg intraperitoneal) was given at baseline and G-CSF was given on days 2, 3, 4, and 5. In cyclophosphamide-bortezomib-G-CSF group (CBG), bortezomib at baseline was added to CG experiment. Results: Bortezomib compared with PBS in B6 mice resulted in a significantly higher CFU-C 12 h to 18 h after baseline injections (mean peak CFU-C: 680/ml vs. 100/ml respectively, fold increase in CFU-C: 6.8 vs. 0.8 respectively, P = 0.0002) (Figure 1). In bortezomib group, CFU-C remained at peak from 12 h to 18 h and returned close to baseline 24 h after bortezomib. White blood cell (WBC) peak of 1.5 fold over baseline was observed 12 h to 15 h after bortezomib. There was no statistically significant difference in bortezomib HSPC mobilization in non-splenectomized vs. splenectomized mice (mean peak of 580/ml vs. 550/ml respectively, P = 0.89). In VLA4KO experiments, there was no statistically significant difference in peak CFU-C in bortezomib group versus PBS group (mean 730/ml vs. 590/ml respectively, P = 0.18) suggesting no HSPC mobilization effect for bortezomib in VLA4KO mice (Figure 2). In bortezomib plus G-CSF (BG) experiments, peak CFU-C on day 6 in BG group was significantly higher than G-CSF group (mean peak CFU-C: 4700/ml vs. 2400/ml respectively, P = 0.005). In G-CSF plus bortezomib (GB) experiments, peak CFU-C on day 6 in GB group was significantly higher than G-CSF group (mean peak CFU-C: 5600/ml vs. 2400/ml respectively, P = 0.001). There was no statistically significant difference in peak CFU-C in BG vs. GB groups (P = 0.28). In bortezomib plus AMD3100 (BA) vs. PBS plus AMD3100 (PA) experiments, peak CFU-C 3 h after AMD3100 in BA group was significantly higher than PA group (peak CFU-C: 2600/ml vs. 1100/ml respectively, fold increase in CFU-C: 105 vs. 12.5 respectively, P = 0.01) (Figure 3). In cyclophosphamide-G-CSF (CG) vs. cyclophosphamide-bortezomib-G-CSF (CBG) chemomobilization experiments, CBG compared to CG resulted in a trend toward higher CFU-C peak on day 6 (23500/ml vs. 21000/ml respectively, P = 0.49) and higher CFU-C on day 7 (14200/ml vs. 8700/ml respectively, P = 0.03). Conclusion: Bortezomib is a potent HSPC mobilizer drug, augment AMD3100 and G-CSF mobilization in at least an additive fashion, and increase and extend chemomobization effect of cyclophosphamide. Bortezomib mobilization mechanism probably involves VLA4/VCAM-1 axis. Bone marrow rather than spleen is the source of HSPC's mobilized by bortezomib. Disclosures: No relevant conflicts of interest to declare.

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EXTRACTION OPTIMIZATION AND ANTIOXIDANT ACTIVITY OF PHENOLIC COMPOUNDS FROM AVOCADO PEEL

Journal of Medicine and Pharmacy ◽

10.34071/mp.2019.3.7 ◽

2019 ◽

pp. 49-59

Author(s):

Nu Linh Giang Ton ◽

Thi Hoai Nguyen ◽

Quoc Hung Vo

Keyword(s):

Antioxidant Activity ◽

Response Surface Methodology ◽

Antioxidant Capacity ◽

Response Surface ◽

Total Antioxidant Capacity ◽

Total Phenolic Content ◽

Phenolic Content ◽

Quadratic Model ◽

Total Phenolic ◽

Total Antioxidant

Avocado peel has been considered as a potential source of natural antioxidants in which phenolics are among the most important compounds. Therefore, this study aims to optimize the extraction process of phenolics using response surface methodology and evaluate the corresponding antioxidant activity. From the quadratic model, the optimal condition was determined including the ethanol concentration 54.55% (v/v), the solvent/solute ratio 71.82/1 (mL/g), temperature 53.03 oC and extraction time 99.09 min. The total phenolic content and the total antioxidant capacity at this condition with minor modifications were 26,74 ± 0,04 (mg GAE/g DW) and 188.06 ± 1.41 (mg AAE/g DW), respectively. The significant correlation between total phenolic content and total antioxidant capacity was also confirmed. Key words: response surface methodology, central composite rotatable design, total phenolic content, total antioxidant capacity, avocado peel

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