Decrease In Number Of Viable Cells Research Articles

Self-assembled elastin-like polypeptide fusion protein coacervates as competitive inhibitors of advanced glycation end-products enhance diabetic wound healing

Chronic and non-healing skin wounds are some of the most significant complications in patients with advanced diabetes. A contributing mechanism to this pathology is the non-enzymatic glycation of proteins due to hyperglycemia, leading to the formation of advanced glycation end products (AGEs). AGEs bind to the receptor for AGEs (RAGE), which triggers pro-inflammatory signals that may inhibit the proliferative phase of wound healing. Soluble forms of RAGE (sRAGE) may be used as a competitive inhibitor of AGE-mediated signaling; however, sRAGE is short-lived in the highly proteolytic wound environment. We developed a recombinant fusion protein containing the binding domain of RAGE (vRAGE) linked to elastin-like polypeptides (ELPs) that self-assembles into coacervates at around 30–31 °C. The coacervate size was concentration and temperature-dependent, ranging between 500 and 1600 nm. vRAGE-ELP reversed several AGE-mediated changes in cultured human umbilical vein endothelial cells, including a decrease in viable cell number, an increase in levels of reactive oxygen species (ROS), and an increased expression of the pro-inflammatory marker, intercellular adhesion molecule-1 (ICAM-1). vRAGE-ELP was stable in elastase in vitro for 7 days. When used in a single topical application on full-thickness excisional skin wounds in diabetic mice, wound closure was accelerated, with 90% and 100% wound closure on post-wounding days 28 and 35, respectively, compared to 62% and 85% on the same days in animals treated with vehicle control, consisting of ELP alone. This coacervate system topically delivering a competitive inhibitor of AGEs has potential for the treatment of diabetic wounds.

Menin‑MLL inhibitors induce ferroptosis and enhance the anti‑proliferative activity of auranofin in several types of cancer cells.

Menin‑mixed‑lineage leukemia (MLL) inhibitors have potential for use as therapeutic agents for MLL‑rearranged leukemia. They are also effective against solid cancers, such as breast cancer. The present study demonstrated that menin‑MLL inhibitors, such as MI‑463, unexpectedly induced the ferroptotic cell death of several cancer cell lines. MI‑463 at a double‑digit nM concentration markedly decreased the viable number of OVCAR‑8 ovarian cancer cells for 3days. Ferrostatin‑1 (a ferroptosis inhibitor) almost completely abrogated the MI‑463‑induced decrease in viable cell numbers. Furthermore, the cancer cell‑killing activity was inhibited by N‑acetylcysteine [a scavenger of reactive oxygen species (ROS)], deferoxamine (DFO, an iron chelator), PD146176 (a specific inhibitor of arachidonate 15‑lipoxygenase), idebenone (a membrane‑permeable analog of CoQ10) and oleic acid [a monounsaturated fatty acid and one of the end products of stearoyl‑CoA desaturase 1 (SCD1)], whereas Z‑VAD‑FMK (an apoptosis inhibitor) had a negligible effect on cell death. It was also found that MI‑463 in combination with auranofin (a thioredoxin reductase inhibitor) synergistically increased cancer the death of breast, ovarian, pancreatic and lung cancer cell lines (88%, 14/16 cell lines). The synergistic induction of cell death was abrogated by ferroptosis inhibitor and DFO. Inhibitors of SCD1, similar to MI‑463, also enhanced cancer cell death synergistically with auranofin, while inhibitors of SCD1 and MI‑463 did not additively induce cell death. Treatment with zinc protoporphyrin‑9, a specific inhibitor of heme oxygenase‑1 (HO‑1), markedly attenuated the cell death induced by MI‑463 plus auranofin. On the whole, these results suggest that the MI‑463‑induced decrease in cell viability may be at least partly associated with the inhibition of SCD1 activity. In addition, the potent induction of HO‑1 contributed to the synergistic effects of MI‑463 plus auranofin. Therefore, menin‑MLL inhibitors, such as MI‑463, in combination with auranofin represent an effective therapeutic approach for several types of cancer via the induction of ferroptosis.

Immunostimulated Arginase II Expression in Intestinal Epithelial Cells Reduces Nitric Oxide Production and Apoptosis

Increased production of nitric oxide (NO) and subsequent local cytotoxicity to mucosal epithelial cells has been proposed as a putative mechanism involved in the development of necrotizing enterocolitis (NEC). Intestinal epithelial cells (IECs) metabolize L-arginine to either nitric oxide (NO) by NO synthase (NOS) or to L-ornithine and urea by arginase. L-ornithine is the first step in polyamine synthesis important for cell proliferation, while NO production can lead to apoptosis. We hypothesized that in IECs immunostimulation increases both NOS and arginase expression, and that arginase activity mitigates NO production and apoptosis. Rat intestinal epithelial cells (rIEC-6) were immunostimulated by either incubation with lipopolysaccharide (LPS) alone for 24 h or by incubation with conditioned media (CM) for 24 h. CM was obtained from RAW 264.7 cells (a macrophage cell line) treated with LPS (E. coli 0127:B8; 1 μg/ml) for 4 h. The rIEC-6 stimulated with LPS or with CM had significantly higher levels of inducible NOS (iNOS) protein, NO production, and arginase II protein than did the control cells. Direct LPS stimulation of rIEC-6 produced a less robust increase in iNOS expression and NO (represented as nitrite percent of control) than did CM stimulation. Inhibition of arginase using Nω hydroxyl-L-arginine (NOHA) further increased stimulated NO production in rIEC-6. Viable cell numbers were significantly lower in CM stimulated cells after 24 h than in controls, and inhibition of arginase activity with NOHA resulted in a further significant decrease in viable cell numbers. We conclude that immunostimulated arginase expression of rIEC-6 cells tempers cytokine-induced iNOS-derived NO production and apoptosis.

Hypoxic proliferation requires EGFR-mediated ERK activation in human pulmonary microvascular endothelial cells.

We have previously shown that hypoxic proliferation of human pulmonary microvascular endothelial cells (hPMVECs) depends on epidermal growth factor receptor (EGFR) activation. To determine downstream signaling leading to proliferation, we tested the hypothesis that hypoxia-induced proliferation in hPMVECs would require EGFR-mediated activation of extracellular signal-regulated kinase (ERK) leading to arginase II induction. To test this hypothesis, hPMVECs were incubated in either normoxia (21% O2, 5% CO2) or hypoxia (1% O2, 5% CO2) and Western blotting was performed for EGFR, arginase II, phosphorylated-ERK (pERK), and total ERK (ERK). Hypoxia led to greater EGFR, pERK, and arginase II protein levels than did normoxia in hPMVECs. To examine the role of EGFR in these hypoxia-induced changes, hPMVECs were transfected with siRNA against EGFR or a scrambled siRNA and placed in hypoxia. Inhibition of EGFR using siRNA attenuated hypoxia-induced pERK and arginase II expression as well as the hypoxia-induced increase in viable cell numbers. hPMVECs were then treated with vehicle, an EGFR inhibitor (AG1478), or an ERK pathway inhibitor (U0126) and placed in hypoxia. Pharmacologic inhibition of EGFR significantly attenuated the hypoxia-induced increase in pERK level. Both AG1478 and U0126 also significantly attenuated the hypoxia-induced increase in viable hPMVECs numbers. hPMVECs were transfected with an adenoviral vector containing arginase II (AdArg2) and overexpression of arginase II rescued the U0126-mediated decrease in viable cell numbers in hypoxic hPMVECs. Our findings suggest that hypoxic activation of EGFR results in phosphorylation of ERK, which is required for hypoxic induction of arginase II and cellular proliferation.

Protective effect of infrared-A radiation against damage induced by UVB radiation in the melan-a cell line

The present work evaluated the infrared-A (IR-A) protective effect using a light-emitting diode (LED) lamp against the cytotoxic effects of ultraviolet B radiation (UVB). Effects on cell viability (Trypan blue assay), DNA damage (comet assay), lipid peroxidation (FOX method), reactive oxygen species production and antioxidant capacity were analyzed in melan-a, a non-tumoral murine melanocytic cell line. To define the doses used in the interaction experiments between IR-A+UVB, dose/response curves were made after exposure to IR-A or UVB. The IR-A dose chosen was 0.8J/cm2 because this dose caused no significant inhibition of proliferation effects and viability decreased. For UVB exposure, a dose of 0.015J/cm2, which showed a decrease in viable cell number by approximately 50% in relation to control until 72h, was selected. For IR-A+UVB, cell proliferation recovery was showed, decreasing DNA damage and lipid peroxide content when compared to UVB alone. Besides, the results obtained for ROS and antioxidant capacity showed that the protection observed was probably not related to decreased oxidative stress. In conclusion, non-thermal IR-A was capable of protecting the melan-a cells from UVB induced damage.

IKZF2, a Novel Target of MSI2 RNA-Binding Protein Plays an Oncogenic Role in Myeloid Leukemia

Deregulated epigenetic program is found in many cancers, and genetic aberrations of histone methyltransferases contribute to transformation in myeloid leukemias. Post-transcriptional regulation in leukemia has recently been highlighted as a novel way for maintaining the leukemia stem cell (LSC) program. We have recently demonstrated that Msi2 is required for LSC function in a murine MLL-AF9 leukemia model. We determined that MSI2 maintains the mixed-lineage leukemia (MLL) self-renewal program by interacting and retaining efficient translation of critical MLL regulated transcription factors including Hoxa9, Myc and Ikzf2. Despite extensive studies implicating Myc and Hoxa9 in leukemia, the role for Ikzf2 in myeloid leukemia is not known.Ikzf2 is a member of the Ikaros transcription factor family and regulates lymphocyte development by controlling regulatory T-cell function. Ikzf2 is highly expressed in Hematopoietic Stem Cells (HSC) and to investigate if Ikzf2 is involved in HSC function, we utilized mice that have a specific deletion of Ikzf2 in the hematopoietic system through the Vav-cre system. We found similar frequencies for different populations in the stem, progenitor and mature cells in the bone marrow of Ikzf2f/f and Ikzf2D/D mice. Colony assays of isolated Lin- Sca1+ c-Kit+ (LSK) cells from Ikzf2f/f and Ikzf2D/D mice resulted in a comparable number of myeloid progenitor colonies. Furthermore, noncompetitive transplant of Ikzf2f/f and Ikzf2D/D bone marrow cells showed similar chimerism after 34 months indicating that I kzf2D/D mice have normal HSC function and hematopoiesis.To interrogate the role of Ikzf2 in acute myeloid leukemia we utilized the MLL-AF9 retroviral transduction model. Intracellular flow cytometry showed that IKZF2 is highly expressed in the LSC population compared to the non-LSCs. We then transduced Ikzf2f/f and Ikzf2D/D LSK cells with MLL-AF9 and found that Ikzf2 deletion results in a ten-fold reduction in colony formation compared to Ikzf2f/f cells. Transplantation of transduced cells results in delayed leukemia progression with reduced disease burden. Secondary transplantation of the initiation experiment exhibited a significant delay in leukemogenesis in the Ikzf2D/D compared to the Ikzf2f/f mice (median survival of 32 and 19.5 days, respectively). The role for Ikzf2 in maintenance was assessed with an inducible puro-creER system, which resulted in 80% decrease in viable cell number within 24hrs of 4-hydroxytamoxifen (4-OHT) treatment. Flow cytometric analysis showed that the Ikzf2-deficient cells had increased apoptosis and differentiation, shown by AnnexinV/7-AAD and Mac1 expression respectively. Furthermore, inducible deletion of Ikzf2 using puro-creER system in vivo revealed that Ikzf2 deletion leads to a delay in leukemia after tamoxifen administration in mice. These results indicate that Ikzf2 is required for both leukemia initiation and maintenance.To determine a role for IKZF2 in human leukemia cells, we performed Ikzf2 knockdown experiments with shRNAs in Kasumi-1, KG1, KCL22 and MOLM13 cells. Ikzf2 depletion resulted in decreased cell growth and increased apoptosis compared to cells infected with scramble shRNA.To determine the mechanism for how IKZF2 controls leukemia cell survival and self-renewal, we performed gene expression profiling of the Ikzf2-deficient Vav-cre LSCs and demonstrated enrichment in signatures for self-renewal loss, increased differentiation, loss of Myc-regulated genes and loss for targets of Hoxa9 and Meis1. Further analysis overlapping our MSI2 HITS-CLIP data and our differentially regulated genes revealed a strong enrichment suggesting that the MSI2 bound targets are transcriptionally regulated by IKZF2. Lastly, ATAC-sequencing of Ikzf2f/f and Ikzf2D/D LSCs revealed alterations in chromatin accessibility that correlated closely with differentially expressed genes. Utilizing the ATAC-seq data we predicted that HOXA9 and MYC sites were significantly altered. We validated that MYC RNA and protein levels were reduced in both murine and human AML cell lines.In contrast to its known tumor suppressor role in hypodiploid B-ALL and T-ALL, these results suggest that Ikzf2 contributes to MLL leukemia cell initiation and maintenance. Thus, we provide evidence that Ikzf2 can regulate c-MYC expression helping in maintaining the stem cell self-renewal program in LSCs. DisclosuresNo relevant conflicts of interest to declare.

English

Listeria monocytogenes is widely distributed in nature and has been isolated from numerous sources such as meat and fermented meat products. This pathogenic microorganism can resist conditions of low pH, low water activity (Aw), high salt (NaCl) concentrations and the presence of sodium nitrite, being able to survive the commercial sausage manufacturing process. The aim of this work was to evaluate the antilisterial activity of a lactic acid bacterium (Lactococcus lactis subsp. lactis PD 6.9) isolated from Italian-style salami in conditions that simulate salami fermentation. L. lactis PD 6.9 produces nisin Z and grows well in pork lean meat broth, a feature that would be useful to compete with food-borne pathogens. The peak of nisin Z production by L. lactis PD 6.9 in pork lean meat broth occurred after 14 h of fermentation, but the inhibitory activity decreased if the producer organism was maintained in stationary phase. When L. lactis PD 6.9 (107CFU ml-1) and Listeria monocytogenes LMA 20 (106CFU ml-1) were co-inoculated in pork lean meat broth, growth of L. lactis PD 6.9 was unaffected. The decrease in viable cell number of Listeria coincided with an increase in bacteriocin activity produced by L. lactis PD 6.9 in pork lean meat broth. Co-culture experiments indicated that L. lactis PD 6.9 was able to control the growth of L. monocytogenes even if the Listeria population was 1000-fold greater than the L. lactis population. These results demonstrate the potential application of L. lactis PD 6.9 in controlling the growth of L. monocytogenes during salami fermentation and its usefulness as a starter culture for fermented sausages. Key words: Listeria monocytogenes, bacteriocins, lactic acid bacteria, co-cultivation, salami.

Copper Tolerance and Biosorption of Saccharomyces cerevisiae during Alcoholic Fermentation.

At high levels, copper in grape mash can inhibit yeast activity and cause stuck fermentations. Wine yeast has limited tolerance of copper and can reduce copper levels in wine during fermentation. This study aimed to understand copper tolerance of wine yeast and establish the mechanism by which yeast decreases copper in the must during fermentation. Three strains of Saccharomyces cerevisiae (lab selected strain BH8 and industrial strains AWRI R2 and Freddo) and a simple model fermentation system containing 0 to 1.50 mM Cu2+ were used. ICP-AES determined Cu ion concentration in the must decreasing differently by strains and initial copper levels during fermentation. Fermentation performance was heavily inhibited under copper stress, paralleled a decrease in viable cell numbers. Strain BH8 showed higher copper-tolerance than strain AWRI R2 and higher adsorption than Freddo. Yeast cell surface depression and intracellular structure deformation after copper treatment were observed by scanning electron microscopy and transmission electron microscopy; electronic differential system detected higher surface Cu and no intracellular Cu on 1.50 mM copper treated yeast cells. It is most probably that surface adsorption dominated the biosorption process of Cu2+ for strain BH8, with saturation being accomplished in 24 h. This study demonstrated that Saccharomyces cerevisiae strain BH8 has good tolerance and adsorption of Cu, and reduces Cu2+ concentrations during fermentation in simple model system mainly through surface adsorption. The results indicate that the strain selected from China’s stress-tolerant wine grape is copper tolerant and can reduce copper in must when fermenting in a copper rich simple model system, and provided information for studies on mechanisms of heavy metal stress.

Mitogen-activated protein kinase phosphatase-1 prevents lipopolysaccharide-induced apoptosis in immature rat intestinal epithelial cells.

Necrotizing enterocolitis is characterized by intestinal inflammation and epithelial barrier dysfunction. Mitogen-activated protein kinase (MAPK) phosphatase (MKP)-1 plays a pivotal role in the feedback control of MAPK signaling, which regulates inflammation and apoptosis. We hypothesized that MKP-1 prevents lipopolysaccharide (LPS)-induced apoptosis in intestinal epithelial cells. Western blot analysis and qPCR were used to assess MKP-1, MAPK (p38, extracellular signal-regulated kinase (ERK), and c-Jun N terminal kinases (JNK)), caspase 3, caspase 9, tumor necrosis factor (TNF)-α, and cyclooxygenase (COX)-2 expression levels in rIEC-6 enterocytes. MKP-1 expression was inhibited using small interfering RNA (siRNA) methodology. Viable cell number was determined using trypan blue exclusion. LPS stimulation led to activation of p38, JNK, and ERK, and induction of MKP-1 mRNA and protein expression. The induction of MKP-1 was associated with a decrease in p38 phosphorylation, and knockdown of MKP-1 prolonged p38 phosphorylation. While LPS stimulation significantly attenuated proliferation of rIEC-6 cells transfected with scramble siRNA, LPS stimulation resulted in a net decrease in viable cell number in cells transfected with MKP-1 siRNA. Following LPS stimulation, MKP-1 knockdown resulted in greater caspase 3 and 9 activities and greater proinflammatory cytokine (TNF-α, COX-2) expression than in cells transfected with scramble siRNA. Our results demonstrate that MKP-1 has a central role in preventing inflammation-induced apoptosis in rIEC-6 enterocytes.

Cytokine‐Induced Arginase Expression in Intestinal Epithelial Cells Reduce Nitric Oxide Production and Apoptosis

Intestinal epithelial cells are involved in disease progression of inflammatory bowel diseases such as necrotizing entercolitis in preterm neonates. Intestinal epithelial cells metabolize arginine to either nitric oxide (NO) by NO synthase (NOS) or to L-ornithine and urea by arginase. L-ornithine is the first step in polyamine synthesis important for cell viability, while NO production can lead to apoptosis. The hypothesis of this study is that in intestinal epithelial cells inflammation increases both NOS and arginase expression, and that arginase activity mitigates NO production and apoptosis. Rat intestinal epithelial cells (rIEC-6) were incubated with conditioned media (CM) from RAW 264.7 cells (a macrophage cell line) treated with LPS (E.coli 0127:B8; 1μg/ml) for 4 hours. rIEC-6 cells incubated with CM had significantly higher levels of iNOS, cleaved caspase-3, and arginase II protein levels than did control cells. Levels of arginase I were not different between controls and CM treated cells. rIEC-6 incubated in CM had significantly greater NO production than did untreated rIEC-6, and NO production in CM-treated cells was further increased when the arginase inhibitor nor-NOHA was added to the media. Viable cell number was significantly lower after 24 hours with CM than in controls, and inhibition of arginase activity with nor-NOHA resulted in a further significant decrease in viable cell numbers. These data suggest that cytokine-induced arginase expression in rIEC-6 cells tempers cytokine-induced iNOS-derived NO production and apoptosis.

Photodynamic antibacterial and antibiofilm activity of RLP068/Cl against Staphylococcus aureus and Pseudomonas aeruginosa forming biofilms on prosthetic material

Prosthetic joint infections (PJIs) are becoming a growing public health concern in developed countries as more people undergo arthroplasty for bone fixation or joint replacement. Because a wide range of bacterial strains responsible for PJIs can produce biofilms on prosthetic implants and because the biofilm structure confers elevated bacterial resistance to antibiotic therapy, new drugs and therapies are needed to improve the clinical outcome of treatment of PJIs. Antimicrobial photodynamic therapy (APDT), a non-antibiotic broad-spectrum antimicrobial treatment, is also active against multidrug-resistant micro-organisms such as meticillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa. APDT uses a photosensitiser that targets bacterial cells following exposure to visible light. APDT with RLP068/Cl, a novel photosensitiser, was studied by confocal laser scanning microscopy (CLSM) to evaluate the disruption of MRSA and P. aeruginosa biofilms on prosthetic material. Quantitative CLSM studies showed a reduction in biofilm biomass (biofilm disruption) and a decrease in viable cell numbers, as determined by standard plate counting, in the S. aureus and P. aeruginosa biofilms exposed to APDT with the photosensitiser RLP068/Cl. APDT with RLP068/Cl may be a useful approach to the treatment of PJI-associated biofilms.

Changes in the phase variant spectra in the populations of lactic acid bacteria under antibiotic treatment

Effect of the antibiotics kanamycin and ampicillin on growth and phase variation of the populations of four strains of lactic acid bacteria (Lactobacillus sp. M67AT, L. casei MB, Enterococcus faecium M, and E. faecium M3185) was studied. The presence of antibiotics in the medium resulted in a dose-dependent decrease in viable cell numbers and in partial or complete substitution of the dominant S variant with the minor Sm and Sb variants. The variants differed in colony morphology, as well as in some physiological, biochemical, biotechnological, and probiotic characteristics. The Sm type variants of all strains exhibited the highest resistance to antibiotics. High production of exopolysaccharides was found in Sb variants of lactobacilli and in S variants of enterococci. The highest antibacterial activity was found in Sm variants of lactobacilli, especially in Lactobacillus sp. M76AT The latter is biotechnologically the most promising strain, since all variants fermented milk yielding the products with uniformly pronounced functional and organoleptic properties. These patterns are of importance for the understanding of the mechanisms of antibiotic resistance and for selection of the variants with desired properties, as well as for quality control of the lactic acid bacteria starter cultures.

S100B as a potential biomarker for the detection of cytotoxicity of melanocytes

Skin irritation is one of the most common adverse reactions in hydroquinone (HQ) and retinoic acid (RA). Although melanocytes have rarely been considered to be involved in skin irritation, RA and particularly HQ could induce melanocyte toxicity, resulting in depigmentation. We chose S100B as a candidate gene for melanocytotoxicity from a genome-wide transcriptional profiling analysis after applying irritant doses of HQ, RA and sodium lauryl sulphate (SLS) to cultures of keratinocytes and/or melanocytes. In this study, the role of S100B on melanocyte viability and cytotoxicity was examined. S100B was detected in melanocytes, but not in keratinocytes or fibroblasts. Melanocytes after treatment with increasing concentrations of HQ, RA, SLS and urushiol showed significant increases in intracellular and extracellular S100B expression with reduced viable cell number and increased release of lactate dehydrogenase. No RAGE expression and no significant function of CD166/ALCAM in melanocyte survival and cytotoxicity favoured the role of intracellular S100B in chemically irritated melanocytes. S100B knock-down increased apoptosis through inhibition of PI3K/AKT, NF-κB and ERK activation, suggesting the increased intracellular S100B expression by chemical irritation as a compensatory reaction to reduce cytotoxicity. The numerical decrease in S100B/c-kit-double-positive melanocytes was also examined in human skin epidermis irritated by HQ or RA with stronger staining intensities of S100B. Collectively, the decrease in viable cell number by reduced intracellular S100B levels in vitro and by chemical irritation in vivo suggests that S100B could be a potential biomarker for melanocytes cytotoxicity.

Combination of AKT inhibition with autophagy blockade effectively reduces ascites-derived ovarian cancer cell viability

Recent genomics analysis of the high-grade serous subtype of epithelial ovarian cancer (EOC) show aberrations in the phosphatidylinositol 3-kinase (PI3K)/AKT pathway that result in upregulated signaling activity. Thus, the PI3K/AKT pathway represents a potential therapeutic target for aggressive high-grade EOC. We previously demonstrated that treatment of malignant ascites-derived primary human EOC cells and ovarian cancer cell lines with the allosteric AKT inhibitor Akti-1/2 induces a dormancy-like cytostatic response but does not reduce cell viability. In this report, we show that allosteric AKT inhibition in these cells induces cytoprotective autophagy. Inhibition of autophagy using chloroquine (CQ) alone or in combination with Akti-1/2 leads to a significant decrease in viable cell number. In fact, Akti-1/2 sensitizes EOC cells to CQ-induced cell death by exhibiting markedly reduced EC50 values in combination-treated cells compared with CQ alone. In addition, we evaluated the effects of the novel specific and potent autophagy inhibitor-1 (Spautin-1) and demonstrate that Spautin-1 inhibits autophagy in a Beclin-1-independent manner in primary EOC cells and cell lines. Multicellular EOC spheroids are highly sensitive to Akti-1/2 and CQ/Spautin-1 cotreatments, but resistant to each agent alone. Indeed, combination index analysis revealed strong synergy between Akti-1/2 and Spautin-1 when both agents were used to affect cell viability; Akti-1/2 and CQ cotreatment also displayed synergy in most samples. Taken together, we propose that combination AKT inhibition and autophagy blockade would prove efficacious to reduce residual EOC cells for supplying ovarian cancer recurrence.

Unraveling The Molecular and Metabolic Basis For Glutamine Addiction In Leukemias

The bone marrow (BM) microenvironment is characterized by hypoxia and the presence of supporting mesenchymal stromal cells (MSC) that promote leukemia cell survival and resistance to therapy, in part by metabolic reprogramming. However mechanisms that couple leukemic cells survival to metabolic processes under different microenvironment conditions have not been elucidated. Glutamine (Gln) provides cells with carbon skeletons to the Krebs cycle (KC) via anaplerosis, sustains cell proliferation, regulates redox homeostasis and modulates activity of signal transduction pathways. Recent data suggests that leukemia cells reduce molecular oxygen utilizing electrons from carbon sources other than pyruvate, and we hypothesize that these electrons could be provided at least in part by glutaminolysis. Our recent studies utilizing gene expression profiling indicate that MSC co-culture under hypoxia promoted glycolytic gene expression in AML cells, as well as genes regulating oxidative phosphorylation (OXPHOS), KC cycle and Gln utilization (GLS1, GOT) (Matre et al., AACR 2013:1887). Here we report studies aimed to unravel metabolic changes in proliferating leukemic cells under hypoxia and upon interaction with MSC and determine the role of Gln as a contributor.First, we performed GC-MS metabolic profiling of OCI-AML3 leukemic cells alone or in co-cultured with MSC under hypoxic or normoxic conditions and observed significant changes in the core metabolic processes. Our data demonstrates that microenvironment promotes glucose-independent OXPHOS to meet bioenergetics needs of leukemic cells. Interaction with MSC propels a glucose-independent oxidative KC through Gln and asparagine catabolism even under conditions where oxygen concentration is limited. Under hypoxia, concentrations of KC intermediates were lower compared to normoxia, however the accumulation of 2-hydroxyglutarate suggests reverse KC activity with glutamate-derived 2-oxoglutarate being converted to citrate via reductive carboxylation pathway. In addition, consumption of glucogenic amino acids was upregulated by MSCs. Glycolytic intermediates accumulated under hypoxia and coculture accompanied by excretion of pyruvate as lactate, suggesting increased availability of carbon skeletons for biomass generation provided, in part, by glutaminolysis.Next, oxygen consumption rates (OCR) and extra-cellular acidification rates (ECAR) in OCI-AML3 and REH cells were assayed using Seahorse Bioscience XF96 EF Analyzer (Billerica, MA). Glutaminase (GLS) inhibition by BPTES or shRNA caused a decrease in basal OCR, reduced ATP production and decreased maximal respiratory capacity of leukemic cells (Fig. 1). Both acute and prolonged exposure to BPTES resulted in a compensatory increase in glycolytic activity as shown by increase in ECAR and confirmed by media lactate levels. [Display omitted] Analysis of a panel of acute leukemia cell lines (n=12) showed that subset of leukemia (75%) markedly dependent on Gln for growth with Gln deprivation causing steep decrease in viable cell number via induction of apoptosis. In addition, in the corresponding subset, inhibition of GLS (GLS1) with BPTES decreased cell growth and increased apoptosis under both normoxia and hypoxia. Notably, MSC co-culture failed to protect firmly attached hypoxic AML cells, which are otherwise resistant to chemotherapy-induced cytotoxicity. Finally, the expression of GLS1 gene splice variants, Glutaminase C (GAC) and kidney glutaminase (KGA), was determined using oligonucleotide microarrays (HG U133 Plus 2.0, Affymetrix) in 288 AML and in 103 normal samples (healthy BM and non-leukemia conditions, Haferlach, JCO 2010). GAC transcript was found to be significantly overexpressed in several AML subtypes, including AML with FLT3 gene mutations and complex cytogenetics. In turn, KGA expression was not different between AML and normal samples.In summary, our results indicate that Gln is a major source of carbon skeletons for KC activity in AML cells, and demonstrate the key role of Gln utilization pathway for the survival of hypoxic BM-resident leukemic cells and “Glutamine-dependent OXPHOS subset” of leukemia. These findings support the notion of targeting microenvironment-fueled leukemia metabolism through pharmacological inhibition of GLS with novel selective GLS1/2 inhibitors entering clinical arena. Disclosures:No relevant conflicts of interest to declare.

Antimir-21 Causes Autophagy in the K562 Cell Line and Sensitizes Cells to the Effect of Doxorubicin and Etoposide

AbstractAbstract 4908 Background:microRNAs (miRs) are short non-coding RNAs that regulate gene expression by binding to targeted mRNAs and causing their degradation or inhibition of translation. Some miRs have been described to play a role in disease pathogenesis and response to therapy in cancer [Lee and Dutta (2009), Annu Rev Pathol 4:199–227]. Other miRs have been associated with cancer drug resistance. For example, miR-21 has been shown to be involved in chemoresistance in a variety of cancer cells, namely leukemia cells [Li et al. (2010), Hematology 15:215–21] and to target several genes known to be involved in apoptosis, such as Pdcd4 [Lu et al. (2008), Oncogene 27:4373–79], FasL [Sayed et al. (2010), J Biol Chem 285:20281–90] and PTEN [Meng et al. (2007), Gastroenterology 133:647–58]. A recent report showed that miR-21 targets and upregulates Bcl-2 [Dong et al. (2011), Arch Med Res 42:8–14] by a non-canonical mechanisms, as demonstrated for other miRs [Lu et al. (2010), Cardiovasc Res 86:410–20]. Given that miR-21 regulates Bcl-2 expression and it has been well established that Bcl-2 has a role in the control of cellular autophagy, the aim of this study was to investigate if antimiR-21 is involved in autophagy in a Chronic Myeloid Leukemia cell line (K562) and if the influence of antimiR-21 in drug sensitivity was associated with autophagy. Methods:K562 cells were transfected with antimiR-21 (or control antimiR) and further treated with etoposide, doxorubicin or with an appropriate control. Additional experiments were carried out by treating cells with 3-MA (inhibitor of autophagy) or serum starvation (inducer of autophagy). RT-qPCR was used to confirm downregulation of miR-21 levels. Cellular proliferation (BrdU assay), cell cycle (flow cytometry following PI labeling) and programmed cell death (TUNEL assay) were analyzed. The expression levels of proteins involved in apoptosis (Bcl-2, PARP) and in autophagy (Beclin-1, Vps34 and LC3B) were investigated (Western Blot). Cellular sensitization to the effects of doxorubicin or etoposide was confirmed by counting viable cell number (Trypan blue). Monodansylcadaverine (MDC) was used to assess the presence of autophagic vacuoles by immunofluorescence microscopy. The presence of autophagosomes was analysed by transmission electron microscopy (TEM). Results:miR-21 expression was downregulated with antimiR-21. Downregulation of miR-21 caused a decrease in viable cell number, no alterations in the cell cycle profile, decreased cellular proliferation and increased programmed cell death. Although Bcl-2 expression was downregulated, PARP cleavage was not affected, indicating that the mechanism of cell death was not apoptotic. The expression levels of autophagy-related proteins such as Beclin-1, Vps34 and LC3B-II was increased and autophagic vacuoles typical of MDC staining were observed. The presence of autophagic vacuoles was further confirmed by TEM. Downregulation of miR-21 with antimiR-21 sensitized K562 cells to the effects of doxorubicin (by 26% comparing to treatment with doxorubicin alone and by 22% comparing to treatment with antimiR-21 alone) and of etoposide (by 23% comparing to treatment with etoposide alone and by 38% comparing to treatment with antimiR-21 alone). This effect was reversed by treatment with the inhibitor of autophagy, 3-MA. Moreover, treatment with 3-MA also reverted the sensitivity of cells to the drugs when used alone (without antimiR-21), indicating that these drugs induced autophagy. In agreement with this, serum starvation, which induces autophagy, also sensitized cells to the effect of these drugs. Conclusion:miR-21 downregulation caused autophagy and sensitized K562 chronic myeloid leukemia cells to doxorubicin and etoposide. Serum starvation sensitized cells to death induced by these drugs, indicating that autophagy is involved in the cell death induced by these drugs. In addition, it was further confirmed that autophagy was involved in antimiR-21 induced sensitization to the drugs, by reverting sensitization of cells to these drugs with an inhibitor of autophagy (3-MA). Acknowledgments:Fundação Calouste Gulbenkian for financial support. FCT for grants to HS (SFRH/BD/47428/2008) and RTL (SFRH/BPD/68787/2010). GMA is supported by FCT and the European Social Fund. IPATIMUP is an Associate Laboratory of the Portuguese Ministry of Science, Technology and Higher Education and is partially supported by FCT. Disclosures:No relevant conflicts of interest to declare.

Effects of HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) on NEU/HER2 overexpressing mammary tumours in MMTV-NEU-NT mice monitored by Magnetic Resonance Spectroscopy

BackgroundThe importance of ERBB2/NEU/HER2 in the response of breast tumours to the heat shock protein 90 (HSP90) inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG; tanespimycin) has been demonstrated in the clinic. ERBB2 is an oncoprotein client that is highly dependent on HSP90. This and other oncogenic client proteins (e.g. B-RAF, C-RAF, ALK and CDK4) are depleted by 17-AAG in both animal tumours and patients. Here we investigate by Magnetic Resonance Spectroscopy (MRS) the metabolic response of 17-AAG in spontaneous, NEU/HER2 driven mammary tumours in transgenic MMTV-NEU-NT mice and in cells isolated and cultured from these tumours.MethodsMammary tumours were monitored by 31P MRS in vivo and in tumour extracts, comparing control and 17-AAG treated mice. A cell line derived from NEU/HER2 mammary tumours was also cultured and the effect of 17-AAG was measured by 31P MRS in cell extracts. Molecular biomarkers were assessed by immunoblotting in extracts from cells and tumours. For comparison of tumour volume, metabolite concentrations and Western blot band intensities, two-tailed unpaired t-tests were used.ResultsThe NEU/HER2 mammary tumours were very sensitive to 17-AAG and responded in a dose-dependent manner to 3 daily doses of 20, 40 and 80mg/kg of 17-AAG, all of which caused significant regression. At the higher doses, 31P MRS of tumour extracts showed significant decreases in phosphocholine (PC) and phosphoethanolamine (PE) whereas no significant changes were seen at the 20mg/kg dose. Extracts of isolated cells cultured from the mammary carcinomas showed a significant decrease in viable cell number and total PME after 17-AAG treatment. Western blots confirmed the expected action of 17-AAG in inducing HSP72 and significantly depleting HSP90 client proteins, including NEU/HER2 both in tumours and in isolated cells.ConclusionsThe data demonstrate the high degree of sensitivity of this clinically relevant NEU/HER2-driven tumour model to HSP90 inhibition by 17-AAG, consistent with the clinical data, and suggest that the metabolic signature of choline phospholipids obtained by MRS could be useful both as a preclinical and clinical tool for investigating surrogate markers of response to treatment.

Effect of highly lipolyzed goat cheese on HL-60 human leukemia cells: Antiproliferative activity and induction of apoptotic DNA damage

To establish cheese as a dairy product with health benefits, we embarked on examining the multifunctional role of cheeses, especially in the field of cancer prevention. The current study was designed to investigate whether different types of commercial goat cheeses may possess antiproliferative activity, using an HL-60 human promyelocytic leukemia cell line as a cancer cell model. Among 11 cheese extracts tested at 500μg/mL, 6 (Crottin de Chavignol, Pouligny Saint-Pierre, Chabichou du Poitou, Valencay, Kavli, and Sainte-Maure de Touraine) resulted in a significant decrease of cell viability, which is consistent with a decrease in viable cell number. Compared with the half-maximal inhibitory concentration (IC50) value of individual cheeses in cellular proliferation assays, the Pouligny Saint-Pierre extract showed strong inhibition. Incubation of cells in the presence of Pouligny Saint-Pierre extract resulted in induction of cellular morphological changes and apoptotic DNA fragmentation as well as expression of the active form of caspase-3 protein. Based on the quantification of the ratio of free fatty acids to triglycerides in different cheese samples, a significant correlation was detected between lipolytic ripeness and IC50 values for antiproliferative capacity tested in HL-60 cells. Collectively, these results support a potential role of highly lipolyzed goat cheeses in the prevention of leukemic cell proliferation.

Clitocybe alexandri extract induces cell cycle arrest and apoptosis in a lung cancer cell line: Identification of phenolic acids with cytotoxic potential

Mushrooms are a possible rich source of biologically active compounds with the potential for drug discovery. The aim of this work was to gain further insight into the cytotoxicity mechanism of action of Clitocybe alexandri ethanolic extract against a lung cancer cell line (NCI-H460 cells). The effects on cell cycle profile and levels of apoptosis were evaluated by flow cytometry, and the effect on the expression levels of proteins related to cellular apoptosis was also investigated by Western blot. The extract was characterised regarding its phenolic composition by HPLC-DAD, and the identified compounds were studied regarding their growth inhibitory activity, by sulforhodamine B (SRB) assay. The effect of individual or combined compounds on viable cell number was also evaluated using the Trypan blue exclusion assay. It was observed that the C. alexandri extract induced an S-phase cell cycle arrest and increased the percentage of apoptotic cells. In addition, treatment with the GI50 concentration (concentration that was able to cause 50% of cell growth inhibition; 24.8μg/ml) for 48h caused an increase in the levels of wt. p53, cleaved caspase-3 and cleaved poly (ADP–ribose) polymerase (PARP). The main components identified in this extract were protocatechuic, p-hydroxybenzoic and cinnamic acids. Cinnamic acid was found to be the most potent compound regarding cell growth inhibition. Nevertheless, it was verified that the concomitant use of the individual compounds provided the strongest decrease in viable cell number. Overall, evidence was found for alterations in cell cycle and apoptosis, involving p53 and caspase-3. Furthermore, our data suggests that the phenolic acids identified in the extract are at least partially responsible for the cytotoxicity induced by this mushroom extract.

적외선 살균이 고춧가루의 품질에 미치는 영향

고춧가루의 살균을 위하여 이송판 상부와 하부에 각각 총 4개의 적외선램프(총출력 8000 W)와 온수순환장치를 장착하고 진동장치를 사용해 분체를 이송하는 분체살균장치를 제작하였다. 진균은 2000 W 이상의 적외선 출력에서 대한 완전살균이 가능하였다. 그러나 일반세균수는 유의적으로 감소하지는 아니하였다. 고춧가루 중 미생물의 효과적인 살균과 수분함량의 감소를 줄이기 위한 고춧가루의 최적 이동속도는 106-164 g/min 범위로 확인되었다. 고춧가루의 입자크기와 무관하게 진균을 완전히 살균할 수 있었으며 수분 함량은 9 mesh보다는 32 mesh의 고운 고춧가루가 더 높은 수분함량의 감소를 보여 주었다. 고춧가루에 대한 반복된 적외선 살균은 고춧가루의 수분함량(초기 12.6%)의 급속한 감소(2회 살균 후 4.6%)로 인하여 바람직하지 아니하였다. 1회 살균만으로도 진균은 불검출 되었으며 살균 횟수가 증가할수록 수분함량이 감소하였다. 한편 적외선살균 후 고춧가루의 총 capsaicinoid의 함량과 ASTA 색도, 표면색도는 모든 살균 조건에서 살균전과 비교하여 유의적인 차이를 보이지 않았다. A tray type-infrared (IR) pasteurization system was developed for decreasing microorganisms in red pepper powder (RPP). The RPP was passed through a tray by a vibrating mode under the 4 IR lamps (total 8000 W) and by circulating water under the tray. Fungi was pasteurized by applying power higher than 2000 W to the RPP. The decrease in viable cell numbers of bacteria, however, was not observed under the same conditions. Conveying speed of RPP was optimized to 106-164 g/min on the basis of microbial reduction and retaining of moisture content of RPP. The water content of 32 mesh-RPP decreased rapidly after pasteurization. However, fungi in both RPPs could be sterilized regardless of particle sizes. The repetition of IR pasteurization was not favourable due to severe decrease of water content in RPP. The IR pasteurization of RPP did not cause significant difference in the capsaicinoid contents, ASTA colour value, and L, a, and b values under all investigated conditions.