Dose-dependent Decrease In Expression Research Articles

Berry Pomace Extracts as a Natural Washing Aid to Mitigate Enterohaemorrhagic E. coli in Fresh Produce.

Enterohemorrhagic Escherichia coli (EHEC) outbreaks have been frequently linked to the consumption of produce. Furthermore, produce grown on organic farms possess a higher risk, as the farmers avoid antibiotics and chemicals. This study sets out to evaluate the effectiveness of advanced postharvest disinfection processes using berry pomace extracts (BPEs) in reducing EHEC load in two common leafy greens, spinach and lettuce. Spinach and lettuce were inoculated with ~5 log CFU/leaf EHEC EDL-933 and then treated with three different concentrations of BPE (1, 1.5, and 2 gallic acid equivalent, GAE mg/mL) for increasing periods of time. After the wash, the bacteria were quantified. Changes in the relative expression of virulence genes and the genes involved in cell division and replication and response against stress/antibiotics were studied. We observed a significant reduction in EHEC EDL933, ranging from 0.5 to 1.6 log CFU/spinach leaf (p < 0.05) washed with BPE water. A similar trend of reduction, ranging from 0.3 to 1.3 log CFU/mL, was observed in pre-inoculated lettuce washed with BPE water. We also quantified the remaining bacterial population in the residual treatment solutions and found the survived bacterial cells (~3 log CFU/mL) were low despite repeated washing with the same solution. In addition, we evaluated the phenolic concentration in leftover BPE, which did not change significantly, even after multiple uses. Alterations in gene expression levels were observed, with downregulation ranging from 1 to 3 log folds in the genes responsible for the adhesion and virulence of EHEC EDL933 and significant upregulation of genes responsible for survival against stress. All other genes were upregulated, ranging from 2 to 7 log folds, with a dose-dependent decrease in expression. This finding shows the potential of BPE to be used for sanitation of fresh produce as a natural and sustainable approach.

Augmentation of 2-arachidonoylglycerol signaling in astrocytes maintains synaptic functionality by regulation of miRNA-30b.

2-Arachidonoylglycerol (2-AG), the most abundant endocannabinoid, displays anti-inflammatory and neuroprotective properties. Inhibition of 2-AG degradation by inactivation of monoacylglycerol lipase (MAGL), a key enzyme degrading 2-AG in the brain, alleviates neuropathology and improves synaptic and cognitive functions in animal models of neurodegenerative diseases. In particular, global inactivation of MAGL by genetic deletion of mgll enhances hippocampal long-term potentiation (LTP) and hippocampus-dependent learning and memory. However, our understanding of the molecular mechanisms by which chronic inactivation of MAGL enhances synaptic activity is still limited. Here, we provide evidence that pharmacological inactivation of MAGL suppresses hippocampal expression of miR-30b, a small non-coding microRNA, and upregulates expression of its targets, including ephrin type-B receptor 2 (ephB2), sirtuin1 (sirt1), and glutamate ionotropic receptor AMPA type subunit 2 (GluA2). Importantly, suppression of miR-30b and increase of its targets by inactivation of MAGL result primarily from inhibition of 2-AG metabolism in astrocytes, rather than in neurons. Inactivation of MAGL in astrocytes prevents miR-30b overexpression-induced impairments in synaptic transmission and long-term potentiation (LTP) in the hippocampus. Suppression of miR-30b expression by inactivation of MAGL is apparently associated with augmentation of 2-AG signaling, as 2-AG induces a dose-dependent decrease in expression of miR-30b. 2-AG- or MAGL inactivation-suppressed expression of miR-30b is not mediated via CB1R, but by peroxisome proliferator-activated receptor γ (PPARγ). This is further supported by the results showing that MAGL inactivation-induced downregulation of miR-30b and upregulation of its targets are attenuated by antagonism of PPARγ, but mimicked by PPARγ agonists. In addition, we observed that 2-AG-induced reduction of miR-30b expression is mediated via NF-kB signaling. Our study provides evidence that 2-AG signaling in astrocytes plays an important role in maintaining the functional integrity of synapses in the hippocampus by regulation of miR-30b expression.

Zebrafish gata2 Orthologs Are Necessary but Not Sufficient for Developmental Hematopoiesis

Germline mutations in the GATA2 gene have been identified in patients with a variety of hematological disorders unified by their with predisposition to myelodysplastic syndromes (MDS) and defective immunity. The GATA2 gene encodes a transcription factor that plays a major role in hematopoiesis. However, the precise effects of GATA2 haploinsufficiency in developmental hematopoiesis and the impact of this in MDS predisposition remain unknown. We have developed zebrafish mutants of gata2 to model this and demonstrate that heterozygosity results in defective primitive and definitive granulopoiesis.Zebrafish carry two orthologues to the mammalian GATA2 gene: gata2a and gata2b . gata2a and gata2b demonstrate distinct expression patterns. By 32 hours post fertilization (hpf), expression of gata2a is seen throughout the dorsal aorta (DA) and cardinal vein, primitive erythrocytes and the nervous system. In contrast, gata2b expression is restricted to the ventral DA (VDA) and branchiomotor neurons. We hypothesised that this division of expression may translate into a requirement for both ohnologs as necessary but not sufficient for normal developmental hematopoiesis.Homozygous gata2a mutants carrying an 11bp deletion (gata2aum27 ) have been previously described. These mutants have abnormal vascular development in the aorta resulting in defective circulation. We analysed developmental hematopoiesis in these mutants. gata2aum27/um27 and gata2aum27 /+ showed normal hemangioblasts, and primitive hematopoiesis before 18 somites. At 22hpf however myeloid transcription factor cebpa showed a dose dependent decrease in expression resulting from gata2a loss, while pu.1 expression remained normal. This suggests that Gata2a may be a direct regulator of cebpa in primitive myeloid cells, or that cebpa expression defines a sub-population of myeloid cells. We then assessed hematopoietic stem cells (HSCs) in gata2aum27 mutants. gata2aum27/um27 showed severely decreased or absent HSCs in the caudal hematopoietic tissue (CHT, fetal liver equivalent) at 52hpf, likely due to absent blood flow in the aorta. gata2aum27/+ mutants also showed a decrease in HSCs at this time point, however this decrease was transient, resolving by 72hpf (A). Further assessment of definitive hematopoiesis showed that gata2um27/um27 lacked mature granulocytes and thymocytes, in keeping with the observed HSC defect. By contrast myeloid cells in gata2aum27/+ at 4dpf (assessed by Sudan Black (SB) stain) were unaffected (B,C) . Interestingly lck-expressing thymocytes showed a range of phenotypes, from normal, through reduced and some with a complete absence of thymocytes in one of their thymii.We next used CRISPR to generate a gata2b mutant line. This line carries a 4bp deletion leading to a truncated protein lacking both zinc fingers (gata2bU5008 ). In contrast to gata2aum27/um27 , gata2bu5008/5008 have normal circulation and were viable beyond 5dpf. Loss of gata2b by morpholino knockdown results in absence of HSC, however gata2b5008/+ showed no difference in HSC numbers in the CHT at 60hpf. Myeloid cells were unaffected in gata2b5008 mutants by SB at 48hpf, however, by 4dpf gata2b5008 show a dose dependent reduction in myeloid cells (D). This suggests that Gata2b regulates myeloid cell proliferation or differentiation after the emergence of HSCs.We next studied haematopoiesis in crosses of gata2aum27/+ to gata2bu5008/+ . Quantification of granulocytes showed normal numbers at 48hpf in all resulting genotypes. By 4dpf gata2b5008/+ have a reduced number of SB expressing granulocytes regardless of their gata2a genotype, suggesting that Gata2a is dispensable for definitive myelopoiesis (E). Quantification of HSCs show HSC numbers are unaffected in gata2aum27/+ ; gata2bu5008/+ at 48hpf. Assessment of thymocytes by contrast shows a greater than additive effect on thymocyte numbers than in single mutants alone, suggesting that both Gata2a and Gata2b contribute to lymphoid development in an additive manner. Further characterisation of the effects of gata2aum27/+ ; gata2bu5008/+ in combination is ongoing.In conclusion our results support the notion at that haploinsufficient levels of Gata2a and Gata2b are necessary but not sufficient for normal developmental hematopoiesis and that combined heterozygous loss of these genes models the haploinsufficieny observed in patients in GATA2 haploinsufficient diseases. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Possible mechanism of bamboo shoots (Bambusa balcooa) induced thyroid disruption - An in vitro study.

Endemic goitre and associated iodine deficiency disorders (IDDs) are a major concern in public health even in the period of post salt iodization in many regions. Among others the consumption of cyanogenic plants found responsible for the persistence of such diseases. Bamboo shoots (BS) is one such cyanogenic plant food that caused disruption of certain thyroid hormone synthesizing regulatory element as has already been reported in our earlier study. In this investigation the possible mechanism of thyrocytes disruption along with interruption of thyroid hormone biosynthesis by BS has been worked out. Commonly consumed BS, Bambusa Balcooa Roxb (BBR) water extract was analysed by GC MS; three doses below IC50 were administered to thyrocytes in culture with and without iodine. Expressions of thyroglobulin (Tg), pendrin (PDS) and monocarboxylate transporter 8 (MCT8) were evaluated in thyrocytes with cell cycle analysis, reactive oxygen species (ROS) generation, DNA oxidation and apoptotic regulation through Bax, Bcl-2 and p53. Phytochemical analysis of BBR extract revealed the presence of precursors and metabolic end products of cyanogenic glycosides. Dose dependent decrease in expression of Tg and PDS with concomitant decrease in gene expression of these with MCT8 were observed. Increased ROS, DNA oxidation and associated imbalance were found through increased Bax and p53 with decreased Bcl-2 that perturbed thyrocytes cell cycle. Cyanogenic constituents of BBR generates ROS associated oxidative changes in thyrocytes with DNA damage and oxidation and cell cycle disruption followed by inhibition of thyroid hormone synthesizing regulatory elements; addition of extra iodine showed partial prevention.

Chlorogenic acid ameliorates experimental colitis in mice by suppressing signaling pathways involved in inflammatory response and apoptosis

Chlorogenic acid (ChA) exhibits a multitude of positive health effects, however, the signaling mechanisms by which ChA could influence the inflammatory response in experimental colitis are unknown. To answer this question, we induced colitis in mice by administration of 2.5% dextran sulfate sodium (DSS) in drinking water for seven days. Oral administration of ChA significantly ameliorated clinical symptoms, improved disease activity index and colon shortening induced by DSS. Furthermore, ChA administration resulted in a suppression of phosphorylated extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Jun N-terminal kinases 1 and 2 (JNK1/2), Akt and signal transducer and activator of transcription 3 (STAT3) with concomitant upregulation of phosphatase and tensin homolog (PTEN) expression. Immunohistochemical analysis showed a dose-dependent decrease in expression and nuclear translocation of nuclear factor-kappa B (NF-κB) p65 subunit, which was accompanied by suppression of pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-α) expression. Induction of apoptosis and oxidative stress was attenuated in a dose-dependent manner by suppressing Bax, caspase-8, caspase-9 and heme oxygenase-1 (HO-1) protein expression in mice administrated with ChA. The results of the current study suggest that ChA could be useful for the treatment of inflammation and attenuating colitis severity by suppressing activation of pro-inflammatory and apoptotic signaling pathways.

EXPRESSION OF BIOLOGICAL MARKERS INDUCED BY IONIZING RADIATION AT THE LATE PERIOD AFTER EXPOSURE IN A WIDE RANGE OF DOSES.

To study radiation induced biological markers of the late period after exposure. A study was performed in 235 Chornobyl accident male clean-up workers exposed in 1986-1987 (doses of external exposure: (M ± SD: 419.48 ± 654.60; range 0.10-3,500 mSv); age 58,34 ± 6,57 years. Controlgroup included 45 non-exposed subjects (mean age: 50.60 ± 5.37 (M ± SD). Gene expression was performed by RT-PCR on 7900HT Analyzer using TLDA for BCL2, CDKN2A, CLSTN2, GSTM1, IFNG, IL1B, MCF2L, SERPINB9, STAT3, TERF1, TERF2,TERT, TNF, TP53, CCND1 genes. Relative telomere length (RTL) was analysed by flow-FISH; immune cell subsets,γ-H2AХ and CyclinD1 expression by flow cytometry. A statistically significant and dose-dependent decrease in expression of the BCL2, SERPINB9, CDKN2A, andSTAT3 genes was demonstrated in parallel to a dose-dependent overexpression of MCF2L and upregulation of TP53 (upto 100 mSv). IL1B expression was the highest in exposed to doses from 0.1 to 100 mSv with a negative correlationbetween at IL1B expression and CD19+3-, CD3-HLA-DR+, CD4+8- cell counts and CD4+/CD8+ ratio. Hyperexpression ofTNF gene in doses above 100 mSv to 1,000 mSv was shown, and in higher doses a combination of TNF downregula-tion with increase in IFNG gene expression were demonstrated with correlations with numbers of CD3+16+56+ andCD25+ lymphocytes and inhibition of expression CLSTN2. An increased expression of γ-H2AХ and Cyclin D1 corre-lated to radiation dose, telomere shortening to age and concommittant pathology. Cellular immunity, gene expression, telomere length, intracellular protein parameters are shown to beamong perspective biological markers at a late period after radiation exposure.

Effects of Histone Deacetylase Inhibitor (Valproic Acid) on the Expression of Hypoxia-inducible Factor-1 Alpha in Human Retinal Müller Cells.

PurposeTo evaluate the effects of valproic acid (VPA), a histone deacetylase inhibitor (HDACI), on the expression of hypoxia-inducible factor-1 alpha (HIF-1α) and vascular endothelial growth factor (VEGF) in human retinal Müller cells under hypoxic conditions.MethodsChemical hypoxia was induced in human retinal Müller cells (MIO-M1) by treatment with increasing concentrations of cobalt(II) chloride (CoCl2). Müller cells were also treated with a set concentration of CoCl2, along with various concentrations of VPA. The expression of HIF-1α and VEGF in the treated Müller cells was determined by enzyme-linked immunosorbent assay.ResultsExposure of human retinal Müller cells to increasing concentrations of CoCl2 produced a dose-dependent increase in HIF-1α expression. The addition of increasing concentrations of VPA lead to a dose-dependent decrease in expression of HIF-1α and VEGF in Müller cells exposed to a set concentration of CoCl2.ConclusionsHDACI VPA downregulated the expressions of HIF-1α and VEGF in human retinal Müller cells under hypoxic conditions. Using HDACI to target HIF-1α expression in Müller cells could be a new therapeutic strategy for the treatment of retinal vascular diseases.

Abstract 2617: SUMOylation inhibition attenuates the cancer stem cell population in glioblastoma and basal breast cancer

Abstract INTRO: TFAP2A and mouse homolog Tcfap2a are transcription factors that play a critical role in development and cancer. We have recently shown that SUMOylation of TFAP2A is a key posttranslational modification that maintains the basal breast cancer phenotype. Treatment with anacardic acid (AA), a SUMOylation inhibitor, led to depletion of the CD44+/hi/CD24-/lo cancer stem cell population. Interestingly, glioblastoma has also been shown to express TFAP2A and demonstrates an expanded cancer stem cell population. METHODS: Mouse 4T1 basal mammary cancer, IOWA-1T human basal breast cancer and UG118 glioblastoma cell lines were analyzed. MTT was used to measure cell viability. Coulter Counter and hemocytometer were used for cell quantification. Matrigel invasion and western blot were used to further characterize the cell lines. RESULTS: Treatment of 4T1 and UG118 cells with AA in vitro resulted in a 67% and 65% decrease in cell viability by MTT assay, respectively (p &amp;lt; 0.05 for both). Quantification of cell number confirmed that AA treatment inhibited cell proliferation. At 48 hours, 4T1 cells demonstrated a decrease of 73-77% (p &amp;lt; 0.01), and UG 118 cells decreased by 97 to 98% (p &amp;lt; 0.001). Cell invasiveness assessed by Matrigel invasion assay was attenuated in 4T1 and UG118 cells after treatment (84%, p &amp;lt; 0.01 and 28%, p &amp;lt; 0.05, respectively). Western blot demonstrated a dose-dependent decrease in expression of the cancer stem cell marker CD44 or the mouse homolog CD49f following AA treatment of UG118 and 4T1 cells, respectively. Western blot confirmed that while total TFAP2A levels were unchanged, SUMO conjugated TFAP2A was decreased after AA treatment of 4T1 cells. To further establish relevance to human cancer, an aggressive chemotherapeutic resistant patient-derived xenograft, IOWA-1T, similarly demonstrated a 41% drop (p &amp;lt; 0.007) in cell invasiveness and a concomitant decrease in SUMOylated TFAP2A with AA treatment. CONCLUSION: Inhibition of the SUMO pathway attenuates the cancer stem cell population in basal breast cancer and glioblastoma. While further investigation is needed, SUMOylation may be a common biological posttranslational modification that defines cancer stem cells. Furthermore, basal breast cancer, brain cancer, and other cancers that express TFAP2A may be amenable to SUMOylation inhibitors such as AA. Citation Format: Jung M. Park, Tong Wu, Sarah Van Dorin, Ronald Weigel. SUMOylation inhibition attenuates the cancer stem cell population in glioblastoma and basal breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2617. doi:10.1158/1538-7445.AM2015-2617

Abstract 5094: Investigating the role of CDC25B in inhibition of cellular proliferation

Abstract CDC25B is a dual specificity phosphatase that activates cyclin-dependent kinases to trigger entry into mitosis and is required for re-entry into mitosis after DNA damage. As an oncogene, CDC25B is overexpressed in multiple tumor types. It has been specifically associated with prostate cancer, where overexpression is seen in prostate tumor tissues and higher levels correlate with increased tumor grade. In the prostate, in addition to its role as a cell cycle regulator, CDC25B acts as an androgen receptor coactivator increasing the response of reporter genes to the addition of androgen analogues. In a transgenic mouse model, overexpression of CDC25B in the mammary glands led to accelerated mammary epithelial proliferation resulting in hyperplasia, while in vitro, co-transfection of CDC25B with Ras allows for transformation of MEF's. Surprisingly, given the establishment of CDC25B as an oncogene, colony formation assays using ectopic expression of CDC25B in transformed cell lines reveal an anti-proliferative effect. Our data reveal a role for Ras inhibition as a cause of this anti-proliferative effect. Co-transfection of CDC25B and Ras results in a dose-dependent decrease in expression of the ectopic Ras protein in several cell lines tested. Preliminary experiments suggest that while mRNA levels remain stable, CDC25B inhibits Ras protein expression. As Ras is pivotal in multiple tumors, this effect of CDC25B may be broadly applicable. These data therefore reveal a new role for CDC25B, which may need to be overcome to allow transformation of tumor cells. Citation Format: Caleb C. Lee, James Manfredi. Investigating the role of CDC25B in inhibition of cellular proliferation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5094. doi:10.1158/1538-7445.AM2014-5094

Abstract 1255: The Mediterranean herb rosemary modulates ER stress proteins to promote androgen receptor degradation in prostate cancer cells

Abstract Background: Prostate cancer is one of the most prevalent forms of cancer among men, especially in the United States. Epidemiological studies have shown that consumption of natural dietary phytochemicals helps in prevention of many types of cancers. One such dietary phytochemical, rosemary (Rosmarinus officinalis), widely used in Mediterranean cuisine is well known for its antitumor activities. However, not much is known about the effect of rosemary on pathogenesis of prostate cancer. Androgens play a critical role in progression of prostate cancer and hence androgen receptor has been a target for prostate cancer treatment since a long time. Herein we have examined the effect of rosemary on prostate cancer progression. Specifically we have examined how rosemary promotes endoplasmic reticulum (ER) stress and modulates the activity and expression of androgen receptor (AR). Materials and Methods: Prostate cancer cell lines, 22Rv1 and LNCaP cells, were used for all experiments. Cell proliferation and cell viability were studied using BrdU and MTT assay respectively. Western blotting, ELISA and siRNA were used to determine the association between ER stress and AR degradation. Results: Rosemary inhibited growth and decreased the cell viability of 22Rv1 and LNCaP cells in a dose-dependent manner. The anti-proliferative effect was associated with apoptosis as evidenced by a significant increase in levels of cleaved caspase-3 protein and TUNEL assay. Moreover, upon treatment with rosemary we saw a dose-dependent decrease in expression of AR in 22Rv1 and LNCaP cells along with decreased PSA levels in LNCaP cells. Rosemary induced apoptosis was associated with ER stress as evidenced by up-regulation of ER stress proteins PERK, IRE1, BiP, CHOP, XBP-1 and caspase- 4. ER stress proteins are known to play an important role in folding of misfolded proteins and degrading continuously misfolded proteins. We therefore explored the relationship between ER stress and AR degradation. Interestingly, we observed that AR degradation was dependent on ER stress proteins BiP and CHOP. Conclusion: Rosemary induces ER stress-mediated apoptotic cell death in prostate cancer cell lines, 22Rv1and LNCaP cells, leading to disruption of AR expression. Our data suggests that this Mediterranean dietary phytochemical possess strong anticancer activity and should be evaluated further as potential chemopreventive and/or chemotherapeutic agent for controlling prostate cancer. Citation Format: Sakina M. Petiwala, Saba Berhe, Gongbo Li, Angela G. Puthenveetil, Larisa Nonn, Jeremy J. Johnson. The Mediterranean herb rosemary modulates ER stress proteins to promote androgen receptor degradation in prostate cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1255. doi:10.1158/1538-7445.AM2014-1255

Tocotrienols suppress non‐small lung cancer cells via downregulation of the Notch‐1 signaling pathway (644.1)

Non‐small cell lung cancer (NSCLC), accounting for 87% of all lung cancers with a 5‐year survival rate of 16%, is the leading cause for global lung cancer deaths. Tocotrienols, isomers of Vitamin E have been shown to exhibit anti‐tumor activity via inhibition of different signaling pathways in tumors. The Notch ‐1 pathway has been reported to be up regulated in lung cancer patients in vivo and in vitro studies. Therefore, the objective of this study was to investigate the interactions and effects of commercially available tocorienol isomers on the Notch‐1 pathway in adenocarcinoma (A549) and squamous cell carcinoma NSCLC (H520) cell lines. Treatment with Tocotrinols resulted in a dose dependent significant decrease in cell growth, cell migration, tumor invasiveness and induction of apoptosis. Molecular mechanisms behind these changes were explored by testing the expression of Notch‐1 and its downstream stream genes by RT‐PCR and western blot analysis. A dose dependent decrease in expression was observed in Notch‐1 and its downstream genes related to proliferation, apoptosis and invasion (Hes‐1, Survivin, PARP, MMP‐9, VEGF, Bcl 2 and Bcl‐XL). In addition, we found a mechanistic link between the Notch‐1 pathway and. NF‐kB. Thus, our data suggests that commercially available Tocotrienols inhibits cell line growth, cell migration, and tumor cell invasiveness via down regulation of Notch 1and NF‐kB pathway while inducing apoptosis, and could therefore be a potential therapeutic approach.

Sour cherry (Prunus cerasus) seed extract increases heme oxygenase-1 expression and decreases proinflammatory signaling in peripheral blood human leukocytes from rheumatoid arthritis patients

Sour cherry seed extract (SCE) was evaluated for its capacity to inhibit lipopolysaccharide-treated human peripheral blood T cells expressing tumor necrosis factor-alpha, and the chemokine interleukin-8. Both proteins are diagnostic biomarkers for inflammatory pathologies. Peripheral blood leukocytes from 11 rheumatoid arthritis (RA) patients and 8 healthy control subjects were co-cultured for 24h in lipopolysaccharide and the extract, then evaluated by flow cytometry for T cell activation and by enzyme-linked immunoassay for lymphocyte-associated heme oxygenase-1 (HO-1) expression. There was a dose-dependent decrease in expression of the immunophenotypes: CD3+TNF-α+, and CD3+IL8+ in cultures from RA patients to a greater extent than in cells from healthy participants. These results suggest that the extract may have a modulatory roll in RA and other inflammatory disorders via the induction of HO-1, thus abating oxidative stress and strengthening regulation of pro-inflammatory signaling pathways.

Abstract 2573: Carnosol and carnosic acid, from rosemary, promote endoplasmic reticulum stress and androgen receptor degradation in prostate cancer cells

Abstract Background: Prostate cancer is one of the most prevalent forms of cancer among men, especially in the United States. Previously, we have shown that carnosol, a dietary diterpene from rosemary (Rosmarinus officinalis), has a unique function in that it is a dual disruptor of androgen and estrogen receptor alpha. Herein, we examined how carnosol and another dietary diterpene from rosemary, carnosic acid, promote endoplasmic reticulum (ER) stress and modulate the activity and expression of androgen receptor (AR). Materials and Methods: Experiments were performed using the prostate cancer cell lines 22Rv1 and LNCaP cells. Cell proliferation and cell viability were studied using BrdU and MTT assay respectively. Western blotting, ELISA and siRNA were used to evaluate the relationship between ER stress and androgen receptor degradation. Results: Both, carnosic acid and carnosol, inhibited growth and decreased the cell viability of 22Rv1 and LNCaP cells in a dose-dependent manner. The anti-proliferative effect was associated with apoptosis as evidenced by a significant increase in levels of cleaved caspase-3 protein. Interestingly, these diterpenes modulated proteins associated with ER stress including BiP, IRE1 and CHOP. Furthermore, we saw a dose-dependent decrease in expression of AR in 22Rv1 and LNCaP cells when treated with either carnosol or carnosic acid. The proteasome has been shown to be regulated by multiple proteins including proteins associated with ER stress. The AR expression increased in presence of proteasome inhibitor, MG-132, suggesting that decrease in AR expression was due to proteasomal degradation. Carnosic acid and carnosol, also showed a decrease in expression of prostate-specific antigen (PSA) levels in a dose-dependent manner. Conclusion: Both, carnosic acid and carnosol, induce ER stress-mediated apoptotic cell death in prostate cancer cell lines, 22Rv1and LNCaP cells, leading to disruption of AR expression. Our data suggests that these natural diterpenes possess strong anticancer activity and should be evaluated further as potential chemopreventive and/or chemotherapeutic agents for controlling prostate cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2573. doi:1538-7445.AM2012-2573

The Potent STAT3/5 Inhibitor, BP-1-102 Demonstrates Significant Anti-Tumor Activity Against Waldenström Macroglobulinemia

Abstract 5101STAT3 is a cytoplasmic transcription factor, transiently activated in response to external stimuli such as growth factors and cytokines. As a transcription factor, STAT3 induces the expression of genes known to be involved in tumorigenesis, implicating STAT3 dysregulation in a number of hallmark oncogenic processes including tumor cell survival, proliferation, angiogenesis, metastasis, and drug resistance. Aberrant STAT3 signaling is prevalent in hematologic malignancies including Waldenstrom Macroglobulinemia (WM), a rare form of B cell non-Hodgkin lymphoma that is characterized by hyper-monoclonal IgM secretion in the peripheral blood. Despite the development of novel therapies and combinatorial treatment regimes, WM remains uniformly fatal, and as the limits of current chemotherapies have been reached, new approaches to treatment are urgently needed to improve patient outcome. Analysis of the gene and protein expression profiles of WM patients suggests that activation of STAT3 signaling plays a critical role in WM, providing rationale for the therapeutic use of STAT3 inhibitors.We have demonstrated pre-clinical efficacy of a novel, highly specific and potent small molecule STAT3/5 inhibitor, BP-1-102, in both hyper-IgM secreting B cell lymphoma cell lines (Mec-1 and RL) as well as two WM cell lines (MWCL-1 and BCWM-1). BP-1-102 directly targets STAT proteins by specifically blocking the SH2 domain that is a required for the phosphorylation, dimerization and nuclear localization of STAT3, ultimately resulting in inhibition of STAT3 transcriptional activation of target genes. We have shown that BP-1-102 directly interacts with STAT3’s SH2 domain and is one of the most effective disruptors of STAT3 activity described to date. Treatment of cell lines with low μM doses of BP-1-102 induced dose-dependent decreases in constitutive and IL10-induced STAT3 phosphorylation (pSTAT3) as well as pSTAT3 nuclear localization. We further evaluated the potency of BP-1-102 against STAT1 and 5 compared to STAT3 using phosphor-flow cytometry to measure STAT phosphorylation status. BP-1-102 effectively inhibited GM-CSF induced STAT5 phosphorylation in AML2 cells at low dose concentrations (< 12.5uM) but only weakly inhibited IFNγ induced STAT1 phosphorylation in U937 leukemic cells at similar dose concentrations confirming the selectivity of BP-1-102 for STAT 3 and 5. Using a STAT3 dependent luciferase reporter construct, we confirmed repression of STAT3 transcriptional activity which correlated with a dose-dependent decrease in expression of STAT3 target genes (Mcl-1, Bcl-XL, Survivin and c-Myc). Inhibition of pSTAT3 resulted in decreased cell viability as assessed by MTT assay after 72 hours of in vitro exposure, with IC50 values ranging from 6uM to 10uM. In addition, treatment of cells with BP-1-102 resulted in caspase-dependent apoptosis which correlated with the activation of caspase-3 and PARP cleavage. Interestingly, co-culture of Mec-1 and RL with bone marrow stroma cells reduced the cytotoxicity of BP-1-102 suggesting stroma-conferred resistance, while MWCL-1 were equally sensitive to the cytotoxic effects of BP-1-102 regardless of either culture condition. Preliminary investigation suggests that the efflux system, used by cells to extrude toxic substances and linked to drug resistance in cancer, may be responsible for conferring stroma-mediate resistance to BP-1-102 in Mec-1 and RL cells. Finally, xenograft experiments to determine in vivo efficacy and safety are planned and will be presented.Collectively, these findings demonstrate a critical role for STAT3 signaling in WM pathology and provide the rationale for further development of STAT3 inhibitors for the treatment of WM. Disclosures:No relevant conflicts of interest to declare.

Quercetin Inhibits IL-1β-Induced Inflammation, Hyaluronan Production and Adipogenesis in Orbital Fibroblasts from Graves' Orbitopathy

Management of Graves' orbitopathy (GO) is challenging, as no reliable, specific, and safe medical therapeutic agents have yet been developed. We investigated the effect of quercetin in primary cultured orbital fibroblasts from GO, targeting pathways of inflammation, aberrant accumulation of extracellular matrix macromolecules, and adipose tissue expansion. Quercetin significantly attenuated intercellular adhesion molecule-1 (ICAM-1), interleukin (IL) -6, IL-8, and cyclooxygenase (COX) -2 mRNA expression, and inhibited IL-1β-induced increases in ICAM-1, IL-6, and IL-8 mRNA. Increased hyaluronan production induced by IL-1β or tumor necrosis factor-α was suppressed by quercetin in a dose- and time-dependent manner. Treatment with noncytotoxic doses of quercetin inhibited accumulation of intracytoplasmic lipid droplets and resulted in a dose-dependent decrease in expression of peroxisome proliferator-activated receptor γ, CCAAT/enhancer-binding protein (C/EBP) α, and C/EBPβ proteins. In conclusion, inhibition of inflammation, hyaluronan production, and adipogenesis by the natural plant product quercetin in vitro provides the basis for further study of its potential use in the treatment of GO.

In vitro anticancer activity of stachydrine isolated from Capparis decidua on prostate cancer cell lines

In this article we report our work on the isolation, characterisation and evaluation of in vitro anticancer activity of stachydrine on solid tumour cells. The in vitro activity was assessed by MTT assay and propidium iodide (PI) staining. Further, an attempt was also made to check the effect of stachydrine on the invasion and metastasis of cancer cells by inhibiting the expression of chemokine receptors (CXCR3 and CXCR4). The influence of stachydrine on the gene expression of CXCR3 and CXCR4 at mRNA and protein levels was examined. Studies revealed a dose dependent decrease in expression of mRNA, and protein levels were observed in stachydrine-treated human prostate cancer cells (PC-3 and LNcaP) as detected by reverse transcriptase-polymerase chain reaction (RT-PCR). The data therefore provides direct evidence for the role of stachydrine as a potent anti-metastatic agent, which can markedly inhibit the malignancy and invasive capacity of malignant cancer cells.

Effects of mycophenolic acid on human bone marrow-derived mesenchymal stem cells in vitro

To investigate the effects of mycophenolic acid (MPA) on the proliferation and differentiation of human bone marrow-derived mesenchymal stem cells (MSCs). MSCs were treated with MPA at the concentration of 1 μ mol/L, 10 μ mol/L, 50 μ mol/L, and 100 μ mol/L, respectively. Cell proliferation was analyzed using CCK-8 method. Apoptosis was detected by PI/Annexin V assay kit. The mRNA expression of inosine-5'-monophosphate dehydrogenase (IMPDH) in MSCs was analyzed by RT-PCR. Osteogenic differentiation was analyzed by Von Kossa staining, Ca(2+) quantification and real-time PCR. In the range of 1 μ mol/L to 100 μ mol/L, MPA caused a significant subdued proliferation rate of MSCs in a concentration-and time-dependent manner by guanosine depletion, and PI/Annexin staining showed no apoptosis induced by MPA. RT-PCR results showed that MSCs expressed both IMPDH I and IMPDH II. von Kossa staining and Ca(2+) quantification indicated that MPA inhibited osteogenic differentiation of MSCs, and real-time PCR detected a dose-dependent decrease in expression of Osteopontin and BMP-2. Further investigation showed that MPA down-regulated the expression of Runx2 and Osterix. MPA can inhibit the proliferation of MSCs by guanosine depletion in a concentration-and time-dependent manner and inhibit the osteogenic differentiation of MSCs by down-regulation of the expression of Runx2 and Osterix.

Abstract P2-09-19: Expression Profiling of a Metastasis Score and a “Glycolytic Index” in Genetically-Defined Human Breast Isogenic Cell Lines Treated with a PI3K Inhibitor

Abstract Background: Activation of signaling pathways, loss of tumor suppressors and oncogenic mutations often lead to dysregulation of glycolysis and tumorigenesis. We performed expression analysis on genetically-defined human isogenic breast cancer cell lines and with PI3K inhibitor exposure to determine impact on a patient-relevant Metastasis Score (MS) (proliferation index) and an assembled “glycolytic index”. A better understanding of the links between cellular metabolism and proliferation may yield insights into tumor cell biology and shed light on potential companion diagnostic and therapeutic opportunities. Methods: A parental MCF10A and three isogenic cell lines harboring K-ras (G12V), PI3Kα (H1047R), and p53 null were grown overnight in the presence of high levels of growth factors in tissue culture flasks. The parental and PI3Kα cells were treated with 0.3 µM, 1 µM,3 µM GDC-0941 or DMSO. All cell lines were incubated for an additional 24 hr. RNA was extracted with RNeasy (Qiagen) and expression analysis performed by RT-PCR. A total of 28 genes were profiled that included 14 genes in a metastasis score (MS) (Tutt et al), 11 glycolysis-associated genes and 3 reference genes. Expression of each gene was calculated using the ΔΔCT method and “summed” with equal weighting to yield an MS and a “glycolytic index”. Results: The MS and phenotypes for the four untreated cell lines were similar under the monolayer culture conditions used. The parental and PI3Kα cells had a similar “glycolytic index” while p53 null and K-ras cells had elevated indices. Treatment of the parental and PI3Kα with GDC-0941, a PI3K inhibitor, showed a dose-dependent decrease in the expression of constituent MS genes and the glycolytic genes as well as the combined scores. The correlation coefficient between MS and the glycolytic index was 0.77. The four genes in MS with the greatest dose-dependent decrease in expression (BUB1, CCNB1, MYBL2 and UBE2S) are associated with CDK1, a seminal participant in cell cycle regulation. The two genes from the “glycolytic index” with the greatest decrease, HK and PKM, are directly involved in glycolysis. The effect of GDC-0941, as reflected by MS and the “glycolytic index” was more pronounced in PI3Kα than the parental cells (p-value=0.0005). Conclusion: The good correlation observed between the MS and “glycolytic index “in the treated cell lines supports an association between proliferation and metabolism upon restriction of cell signal transduction. The dose-dependent expression decreases in the two indices and their constituent genes, suggest that this PI3K inhibitor successfully perturbs both fundamental processes of the cell. Given the ultimate targeting of proliferation suppression, these scores may serve as useful tools in evaluating therapeutic agents, independent of the specific intended pathways. The similar proliferation levels observed in untreated MCF10A and three mutated cell lines suggest either that single oncogenic mutations are insufficient to affect MS or that the in vitro conditions are masking the metastatic/invasive phenotype. Studies are underway to evaluate cells grown in 3D Matrigel that more accurately mimic the tumor microenvironment. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P2-09-19.

Transcriptional analysis of butanol stress and tolerance in Clostridium acetobutylicum.

The effects of challenges with low (0.25%, vol/vol) and high (0.75%) concentrations of butanol on the growth, glucose metabolism, product formation, and transcriptional program of the solvent-tolerant Clostridium acetobutylicum strain 824(pGROE1) and the plasmid control strain 824(pSOS95del) were used to study solvent tolerance and stress response. Strain 824(pGROE1) was generated by groESL overexpression. The growth of 824(pGROE1) was less inhibited than that of 824(pSOS95del), and 824(pGROE1) was able to metabolize glucose over the entire course of the culture (60 h postchallenge) while glucose metabolism in 824(pSOS95del) lasted 24 h. A comparison of their respective DNA array-based transcriptional profiles identified genes with similar expression patterns (these genes are likely to be part of a general butanol stress response) and genes with opposite expression patterns (these genes are likely to be associated with increased tolerance to butanol). Both strains exhibited a butanol dose-dependent increase in expression of all major stress protein genes, including groES, dnaKJ, hsp18, and hsp90; all major solvent formation genes, including aad, ctfA and -B, adc, and bdhA and -B (an unexpected and counterintuitive finding); the butyrate formation genes (ptb and buk); the butyryl coenzyme A biosynthesis operon genes; fructose bisphosphate aldolase; and a gene with homology to Bacillus subtilis kinA. A dose-dependent decrease in expression was observed for the genes of the major fatty acid synthesis operon (also an unexpected and counterintuitive finding), several glycolytic genes, and a few sporulation genes. Genes with opposite expression kinetics included rlpA, artP, and a gene encoding a hemin permease. Taken together, these data suggest that stress, even when it derives from the solvent product itself, triggers the induction of the solvent formation genes.

Reciprocal regulation of expression of the human adenosine 5'-triphosphate binding cassette, sub-family A, transporter 2 (ABCA2) promoter by the early growth response-1 (EGR-1) and Sp-family transcription factors.

The human ABCA2 transporter gene encodes a member of a large family of ATP-binding proteins that transport a variety of macromolecules across biological membranes. We have performed luciferase reporter gene assays with promoter constructs comprising the 5'-flanking region to identify cis-regulatory DNA elements and have mapped the minimal promoter region to 321 bp upstream of the translation start site. We have discovered a functional role for two GC-boxes located in the proximal promoter of the ABCA2 gene that contain overlapping sites for the EGR-1 and Sp1 transcription factors. We observed that oligonucleotides containing overlapping EGR-1/Sp1 sites bind the Sp1, Sp3 and Sp4 transcription factors. When BE(2)-M17 cells were treated with phorbol 12-myristate 13-acetate, we observed inducible expression and binding of the EGR-1 transcription factor to the two GC-boxes. Transfection of Sp1, Sp3 or Sp4 expression constructs into Drosophila S2 induced a dose-dependent increase in transcriptional activation of the ABCA2 promoter, but transfection of EGR-1 alone failed to activate transcription. When increasing amounts of EGR-1 were transfected into the BE(2)-M17 neuroblastoma cells we observed a dose-dependent decrease in expression of the ABCA2 promoter, although expression of the endogenous ABCA2 gene increased following transfection of EGR-1.