Trypan Blue Dye Exclusion Research Articles

In Vitro and In Vivo Cytogenotoxic Effects of Hot Aqueous Extract of Achyrocline satureioides (Lam.) DC.

In this work we extend the toxicological studies of hot aqueous extract of A. satureioides (As-HAE) evaluating cytotoxic and apoptotic effects on human peripheral blood mononuclear cells (PBMCs). We also determine genotoxic action of this extract in vivo. In addition, the extract was chemically characterized. Finally, we established a comparison with previous data of cold aqueous extract. The As-HAE induced cytotoxicity on PBMCs determined by trypan blue dye exclusion (CC50 = 653 μg/mL) and MTT (CC50 = 588 μg/mL) assays being more toxic than cold extract. However, As-HAE as well as cold extract did not induce apoptosis measured by Hoechst 33258 staining, TUNEL assay, and DNA fragmentation analysis. The in vivo micronucleus test showed that As-HAE exerted cytogenotoxic effects on bone marrow of mice, contrary to what was observed with cold extract. The chemical study of As-HAE allowed identifying the flavonoids found in cold extract: luteolin, quercetin, and 3-O-methylquercetin, but at higher concentrations. We suggest that toxic effects induced by As-HAE could be due to high concentrations of these flavonoids. Given that As-HAE is the most used in folkloric medicine, its administration should be controlled in order to prevent potential cell damage.

Light-emitting diode-generated red light inhibits keloid fibroblast proliferation.

Red light is part of the visible light spectrum that does not generate DNA adducts associated with skin cancer and photoaging and may represent a safer therapeutic modality for treatment of keloid scars and other fibrotic skin diseases. Our laboratory previously demonstrated that light-emitting diode-generated red light (LED-RL) inhibits proliferation of skin fibroblasts. The effects of LED-RL on keloidal skin are not well characterized. To determine the effect of LED-RL on keloid-derived fibroblast proliferation and viability in vitro. Irradiation of primary keloid-derived human skin fibroblasts using LED-RL panels was performed in vitro, and modulation of proliferation and viability was quantified using trypan blue dye exclusion assay. Statistical analysis was performed using analysis of variance to compare treatment arms and the Student t-test to compare each treatment arm with the paired bench control arm. Keloid fibroblasts treated with LED-RL 240, 320, and 480 J/cm demonstrated statistically significant dose-dependent decreases in relative proliferation rate of 12.4%, 16.5%, and 28.9%, respectively, compared with matched nonirradiated controls (p < .05) and did not significantly alter viability relative to the matched nonirradiated controls. Light-emitting diode-generated red light can inhibit keloid fibroblast proliferation in a dose-dependent manner without altering viability. Light-emitting diode-generated red light has the potential to contribute to the treatment of keloids and other fibrotic skin diseases and is worthy of further translational and clinical investigation.

Effects of Ixeris dentata Extract on Radical Oxygen Species and Bcl - 2 Family in Human Breast Cancer Cells

The aim of the study was to determine the effects of Ixeris dentata extract (IDE) on anticancer activity in human breast cancer MDA-MB-231 cells at both cellular and molecular levels. The cells were cultured in the presence of 0, 20, 30 and 40 μg/mL Ixeris dentata extract for 24 hours, respectively. At the end of culture, cytochemical analyses for MTT activity, trypan blue dye exclusion, Annexin V-FITC Apoptosis, and radical oxygen species (ROS) were conducted. RT-PCR was also performed to determine whether or not alterations in cell viability affect the Bax/Bcl-2 ratio. MTT assay showed that relative cell viability decreased in a dose-dependent manner (p<0.05). Reduction of cell viability matched well with increased cell membrane permeability as determined by trypan blue dye exclusion test (p<0.05). The rates of intracellular ROS also increased in a similar manner to those of TB-stained cells. There was an associated shift of apoptotic cells from early to late apoptosis between the 30 and 40 μg/mL. Bax/Bcl-2 ratio significantly increased along with significant decreases in Bcl-2 expression between 30 and 40 μg/mL groups (p<0.05). In conclusion, anticancer activity of Ixeris dentata extract is modulated by a reduction in cell viability along with increased membrane permeability, leading to ROS accumulation within cells, and subsequently cell death through an apoptotic pathway that involves Bax and Bcl-2 in human breast cancer MDA-MB-231 cells.

Genotoxic testing of titanium dioxide anatase nanoparticles using the wing-spot test and the comet assay in Drosophila

Titanium dioxide nanoparticles (TiO2 NPs) are widely used for preparations of sunscreens, cosmetics, food and personal care products. However, the possible genotoxic risk associated with this nano-scale material exposure is not clear, especially in whole organisms. In the present study, we explored the in vivo genotoxic activity of TiO2 NPs as well as their TiO2 bulk form using two well-established genotoxic assays, the wing spot test and the comet assay in Drosophila melanogaster. To determine the extent of tissue damage induced by TiO2 NPs in Drosophila larvae, the trypan blue dye exclusion test was also applied. Both compounds were supplied to third instar larvae by ingestion at concentration ranging from 0.08 to 1.60mg/mL. The results obtained in the present study indicate that TiO2 NPs can reach and induce cytotoxic effects on midgut and imaginal disc tissues of larvae, but they do not promote genotoxicity in the wing-spot test of Drosophila. However, when both nano- and large-size forms of TiO2 were evaluated with the comet assay in Drosophila hemocytes, a significant increase in DNA damage, with a direct dose-response pattern, was observed for TiO2 NPs. The results obtained with the comet assay suggest that the primary DNA damage associated with TiO2 NPs exposure in Drosophila could be associated with specific physico-chemical properties of nano-TiO2, since no effects were observed with the bulk form. This study remarks the usefulness of using more than one genetic end-point in the evaluation of the genotoxic potential of nanomaterials.

P-Cresol affects reactive oxygen species generation, cell cycle arrest, cytotoxicity and inflammation/atherosclerosis-related modulators production in endothelial cells and mononuclear cells.

AimsCresols are present in antiseptics, coal tar, some resins, pesticides, and industrial solvents. Cresol intoxication leads to hepatic injury due to coagulopathy as well as disturbance of hepatic circulation in fatal cases. Patients with uremia suffer from cardiovascular complications, such as atherosclerosis, thrombosis, hemolysis, and bleeding, which may be partly due to p-cresol toxicity and its effects on vascular endothelial and mononuclear cells. Given the role of reactive oxygen species (ROS) and inflammation in vascular thrombosis, the objective of this study was to evaluate the effect of p-cresol on endothelial and mononuclear cells.MethodsEA.hy926 (EAHY) endothelial cells and U937 cells were exposed to different concentrations of p-cresol. Cytotoxicity was evaluated by 3-(4,5-Dimethylthiazol-2-yl)-2,5 -diphenyltetrazolium bromide (MTT) assay and trypan blue dye exclusion technique, respectively. Cell cycle distribution was analyzed by propidium iodide flow cytometry. Endothelial cell migration was studied by wound closure assay. ROS level was measured by 2′,7′-dichlorofluorescein diacetate (DCF) fluorescence flow cytometry. Prostaglandin F2α (PGF2α), plasminogen activator inhibitor-1 (PAI-1), soluble urokinase plasminogen activator receptor (suPAR), and uPA production were determined by Enzyme-linked immunosorbant assay (ELISA).ResultsExposure to 100–500 µM p-cresol decreased EAHY cell number by 30–61%. P-cresol also decreased the viability of U937 mononuclear cells. The inhibition of EAHY and U937 cell growth by p-cresol was related to induction of S-phase cell cycle arrest. Closure of endothelial wounds was inhibited by p-cresol (>100 µM). P-cresol (>50 µM) also stimulated ROS production in U937 cells and EAHY cells but to a lesser extent. Moreover, p-cresol markedly stimulated PAI-1 and suPAR, but not PGF2α, and uPA production in EAHY cells.Conclusionsp-Cresol may contribute to atherosclerosis and thrombosis in patients with uremia and cresol intoxication possibly due to induction of ROS, endothelial/mononuclear cell damage and production of inflammation/atherosclerosis-related molecules.

Influence of the polymer amount on bioactivity and biocompatibility of SiO2/PEG hybrid materials synthesized by sol-gel technique.

SiO2/PEG organic-inorganic hybrid materials, which differ in polyethylene glycol (PEG) content, were synthesized by sol-gel technique and the characterization of their structure and biological properties was carried out in order to evaluate the possible use in biomedical field. FT-IR spectroscopy detected that the two components of the hybrids (SiO2 and PEG) are linked by hydrogen bonds between the Si-OH groups of the inorganic phase and the terminal alcoholic groups and/or the ethereal oxygen atoms in the repeating units of polymer. X-ray diffraction analysis ascertained the amorphous nature of the gels and the observation of their morphology by SEM microscopy confirmed that the interpenetration of the two phases (organic and inorganic) occurs on nanometric scale. The biological characterization was carried out as a function of the polymer amount to study its influence on material behavior. The results showed that the synthesized materials were bioactive and biocompatible. The formation of a hydroxyapatite layer, indeed, was observed on their surface by SEM/EDX analysis after soaking in simulated body fluid. Moreover, the biocompatibility of SiO2/PEG hybrids was assessed performing MTT and SRB cytotoxicity tests on fibroblast cell NIH 3T3 after 24 and 48h of exposure, as well as Trypan Blue dye exclusion test. The response to the presence of the investigated materials was positive. The cell growth and proliferation showed dependence on polymer amount and time of exposure to the material extracts. Therefore, the obtained results are encouraging for the use of the obtained hybrids in dental or orthopedic applications.

Targeting the Wnt/β-Catenin Signaling Pathway in Multiple Myeloma: A Possible New Therapeutic Approach to Overcome Bortezomib-Resistance

Multiple myeloma (MM) is a neoplasm of plasma cells that is often fatal, despite the use of high dose chemotherapy and hematopoietic stem cell transplantation. Although new therapeutic approaches, including novel agents such as thalidomide, lenalidomide, and bortezomib (a proteasome inhibitor), are now used clinically and have improved the outcome of patients with MM, most patients eventually relapse, and it remains an incurable disease.Wnt/β-catenin signaling plays a critical role in both cell proliferation and differentiation in normal tissue. β-catenin, a key player and downstream effector in canonical Wnt signaling, is involved in the regulation of cell fate, proliferation, and self renewal of stem cells. The activation of Wnt signaling is aberrantduring the pathogenesis of various malignant neoplasms, and it has been suggested that Wnt/β-catenin signaling is involved in the regulation of cancer stem cells. When the Wnt signaling pathway is activated, stabilized β-catenin translocates to the nucleus, where it interacts with T-cell factor, followed by transcription of target genes such as c-myc, cyclin D1 and survivin. Nuclear β-catenin also recruits the co-activator, cyclic AMP response element-binding protein (CBP), which is essential for hematopoietic cell proliferation and hematopoietic stem cell self renewal, or its related homolog p300, which is involved in hematopoietic cell differentiation. Recent studies have shown that CBP and p300 have distinct functions in the regulation of β-catenin expression: CBP promotes β-catenin expression, whereas p300 inhibits β-catenin expression. In the context of hematological malignancies, β-catenin is overexpressed in myeloma-derived cell lines and primary myeloma cells, whereas expression is very low in normal plasma cells. Myeloma cells are maintained by several growth factors and cytokines, including Wnt ligands secreted by stromal cells in the bone marrow. These data suggest that Wnt/β-catenin signaling contributes to the pathogenesis of MM and thus might be a promising target for the treatment of this incurable hematological malignancy. Moreover, it has been reported that CBP, rather than p300, plays an important role in the expression of apurinic endonuclease/redox factor-1, an important regulator of multidrug resistance, in retinoic acid-induced chemo-resistant myeloma cells, suggesting that CBP is involved in the acquisition of drug resistance. Taken together with the previous data, Wnt/β-catenin signaling, especially CBP, might be an attractive target for new therapeutic agents against MM. ICG-001, small-molecule inhibitor of the canonical Wnt signaling pathway, specifically binds to CBP, thereby disrupting CBP/β-catenin interaction. In the present study, we investigated the role of Wnt/β-catenin signaling in myeloma cells using ICG-001. MTS and trypan blue dye exclusion assays showed that ICG-001 inhibits the proliferation of U266, RPMI8226, and KMS myeloma cell lines in a dose- (0-15 μM) and time- (0-72 h) dependent manner. Assays for apoptotic cell death were performed to determine the cause of growth inhibition by ICG-001 and demonstrated that ICG-001 induced both early and late apoptosis in myeloma cells. To investigate the molecular mechanisms of ICG-001-induced cell death in myeloma cells, the expression of various cell-death associated proteins and down-stream molecules of Wnt/β-catenin signaling was examined. Western blotting analysis showed that ICG-001 arrested cell growth and induced apoptotic cell death in myeloma cells by reducing the expression of three β-catenin target molecules: survivin, cyclin D1, and c-Myc. We next examined the effects of ICG-001 on bortezomib (BTZ)-resistant MM cells. BTZ resistance is an urgent issue in clinics, and therapeutic approaches for overcoming BTZ resistance are important. Interestingly, ICG-001 inhibited the proliferation of both BTZ-sensitive wild-type KMS and BTZ-resistant KMS cells, suggesting that targeting CBP may overcome BTZ-resistance. Furthermore, when combined with cyclosporine, which inhibits non-canonical Wnt/β-catenin signaling, ICG-001 synergistically induced the growth arrest of myeloma cells by inducing apoptotic cell death. These results indicate that inhibition of Wnt/β-catenin signaling may be an attractive therapeutic option both for patients with newly diagnosed MM and for those in a refractory or relapsed state. DisclosuresNo relevant conflicts of interest to declare.

Genotypic and phenotypic characterization of Pseudomonas aeruginosa isolates from post-cataract endophthalmitis patients

Endophthalmitis caused by Pseudomonas aeruginosa is associated with rapid disease progression and poor visual outcome due to high virulence of the organism. Our aim was to characterize the virulence determinants of P. aeruginosa causing post-operative endophthalmitis. Repetitive sequence analysis (ERIC PCR) was done to study the clonal relatedness of the 17 P. aeruginosa isolates. Type 3 secretion system (T3SS) genotypes were determined and the isolates were further classified as invasive or cytotoxic based on gentamicin survival, trypan blue dye exclusion and MTT assays. Phenotypically, the strains were characterized based on bacterial motility patterns, biofilm formation, phospholipase production and antibiotic susceptibility patterns. Most of our ocular isolates were invasive in nature and nearly half of them were multi-drug resistant. 47% of the isolates formed a strong biofilm, whereas the rest formed moderate to weak biofilms, which may account for an increased colonization and antibiotic resistance. Although the T3SS genotypes correlated well with the invasive/cytotoxic nature of the strains, none of the genotypes were associated with any particular phenotypic trait. To the best of our knowledge, this is the first report on the phenotypic characteristics of P. aeruginosa strains causing post-operative endophthalmitis. Our findings demonstrate that these strains have higher invasive potential and an ability to form biofilm which possibly contributes to an increased ocular virulence.

TiO2/PCL hybrid materials synthesized via sol-gel technique for biomedical applications.

The aim of the present work has been the synthesis of organic/inorganic hybrid materials based on titanium dioxide and poly(ε-caprolactone) (PCL) to be used in the biomedical field. Several materials have been synthesized using sol-gel methods by adding different amounts of polymer to the inorganic sol. The obtained gels have been characterized using Fourier transform infrared (FT-IR) spectroscopy, scanning electron microscopy (SEM) and atomic force microscopy (AFM). The FT-IR data allowed us to hypothesize that the structure formed was that of an interpenetrating network, realized by hydrogen bonds between TiOH groups in the sol-gel intermediate species and carbonyl groups in the polymer repeating units. SEM and AFM analyses highlighted that the obtained materials were nanostructurated hybrids. To evaluate the biological properties of the hybrids, their bioactivity and cytotoxicity were investigated as a function of the PCL amount. The bioactivity of the synthesized systems was proven by the formation of a hydroxyapatite layer on the surface of samples soaked in a fluid simulating human blood plasma (SBF). MTT cytotoxicity tests and Trypan Blue dye exclusion tests were carried out exposing NIH-3T3 mouse embryonic fibroblasts for 24 and 48h to extracts from the investigated hybrid materials. The results showed that all the hybrids had a non-cytotoxic effect on target cells.

Auranofin, an anti-rheumatic gold compound, modulates apoptosis by elevating the intracellular calcium concentration ([ca2+]I) in mcf-7 breast cancer cells.

Auranofin, a transition metal complex is used for the treatment of rheumatoid arthritis but is also an effective anti-cancer drug. We investigate the effects of Auranofin in inducing cell death by apoptosis and whether these changes are correlated to changes of intracellular calcium concentration ([Ca2+]i) in breast cancer cells (MCF-7). Cytotoxicity of Auranofin was evaluated using MTS assay and the Trypan blue dye exclusion method. With fluorescent dyes SR-FLICA and 7-AAD apoptotic death and necrotic death were differentiated by Flow cytometry. A concentration dependent decrease in the viability occurred and cells were shifted to the apoptotic phase. Intracellular calcium ([Ca2+]i) was recorded using florescence microscopy and a calcium sensitive dye (Fluo-4 AM) with a strong negative correlation (r = −0.713) to viability. Pharmacological modulators 2-APB (50 μM), Nimodipine (10 μM), Caffeine (10 mM), SKF 96365(20 μM) were used to modify calcium entry and release. Auranofin induced a sustained increase of [Ca2+]i in a concentration and time dependent manner. The use of different blockers of calcium channels did not reveal the source for the rise of [Ca2+]i. Overall, elevation of [Ca2+]i by Auranofin might be crucial for triggering Ca2+-dependent apoptotic pathways. Therefore, in anti-cancer therapy, modulating [Ca2+]i should be considered as a crucial factor for the induction of cell death in cancer cells.

Role of resveratrol on the cytotoxic effects and DNA damages of iododeoxyuridine and megavoltage radiation in spheroid culture of U87MG glioblastoma cell line.

The purpose of this study was to evaluate the effect of resveratrol on cytogenetic damages of iododeoxyuridine (IUdR) and x-ray megavoltage radiation (6 MV) in spheroid model of U87MG glioblastoma cancer cell line using clonogenic and alkaline comet assay. Cells were cultured as spheroids (350 µm) that were treated with 20 μM resveratrol, 1 μM IUdR and 2 Gy of 6 MV x-ray. After treatment, viability of the cells, colony forming ability and the induced DNA damages were examined using trypan blue dye exclusion, colonogenic and alkaline comet assay, respectively. Our results showed that resveratrol could significantly reduce the colony number and induce the DNA damages of the cells treated with IUdR in combination with 6 MV x-ray radiation. That results indicated that resveratrol as an inhibitor of hypoxia inducible factor 1 alpha (HIF-1α) protein in combination with IUdR as a radiosensitizer enhanced the radiosensitization of glioblastoma spheroid cells.

Daily application of low magnitude mechanical stimulus inhibits the growth of MDA-MB-231 breast cancer cells in vitro.

IntroductionMechanical loads can regulate cell proliferation and differentiation at various stages of development and homeostasis. However, the extension of this regulatory effect of mechanical loads on cancer cells is largely unknown. Increased physical compliance is one of the key features of cancer cells, which may hamper the transmission of mechanical loads to these cells within tumor microenvironment. Here we tested whether brief daily application of an external low magnitude mechanical stimulus (LMMS), would impede the growth of MDA-MB-231 aggressive type breast cancer cells in vitro for 3 wks of growth.MethodsThe signal was applied in oscillatory form at 90 Hz and 0.15 g, a regimen that would induce mechanical loads on MDA-MB-231 cells via inertial properties of cells rather than matrix deformations. Experimental cells were exposed to LMMS 15 min/day, 5 days/week in ambient conditions while control cells were sham loaded. Cell proliferation, viability, cycle, apoptosis, morphology and migration were tested via Trypan Blue dye exclusion, MTT, PI, Annexin V, Calcein-AM and phalloidin stains and scratch wound assays.ResultsCompared to sham controls, daily application of LMMS reduced the number and viability of cancerous MDA-MB-231 cells significantly after first week in the culture, while non-cancerous MCF10A cells were found to be unaffected. Flow cytomety analyses suggested that the observed decrease for the cancer cells in the LMMS group was due to a cell cycle arrest rather than apoptosis. LMMS further reduced cancer cell circularity and increased cytoskeletal actin in MDA-MB-231 cells.ConclusionCombined, results suggest that direct application of mechanical loads negatively regulate the proliferation of aggressive type cancer cells. If confirmed, this non-invasive approach may be integrated to the efforts for the prevention and/or treatment of cancer.

Abstract 326: βIII-Tubulin and cell survival: Novel role in endoplasmic reticulum stress and autophagy in non-small cell lung cancer

Abstract Non-small Cell Lung Cancer (NSCLC) survival rates are dismal and chemotherapy resistance is a significant barrier to effective treatment. βIII-Tubulin is aberrantly expressed in a range of cancers, including NSCLC1. Elevated βIII-tubulin expression is associated with chemotherapy resistance and tumor aggressiveness in this disease. Growing evidence links βIII-tubulin as a survival factor in cancer, where it may support tumor cells in the harsh tumor microenvironment by regulating cellular stress responses2. This study aimed to investigate the role of βIII-tubulin in cellular survival and stress responses to glucose starvation in NSCLC cells. METHODS: Previously described2 NCI-H460 cells with stable expression of βIII-tubulin shRNA (βIIIsh4 and βIIIsh59) or a non-targeting control shRNA (Ctrlsh1) were cultured in glucose-free media. Cell viability and proliferation were monitored over 14 days using Trypan Blue Dye Exclusion and Annexin-V Apoptosis assays. Metabolic activity was measured using the XF24 Extracellular Flux Analyzer. GRP78 protein expression was determined by western blotting. XBP-1 splicing assays were used to detect Endoplasmic Reticulum (ER) stress induction. Autophagic activity was examined using western blotting and immunofluorescence staining for LC3B. RESULTS: Our results demonstrated that βIII-tubulin expression affects metabolic activity. Basal rates of glycolytic metabolism were higher in βIII-tubulin knockdown cells compared with control cells (p&amp;lt;0.05). Glucose depletion led to decreased cell number and viability in the βIII-tubulin knockdown cells (p&amp;lt;0.001) and this was supported by increased cell death in two independent βIII-tubulin shRNA clones (βIIIsh4 61.49±1.86%, βIIIsh59 54.43±1.28% viability relative to Ctrlsh1 cells, p&amp;lt;0.05). Moreover, βIII-tubulin knockdown markedly decreased GRP78 levels, a master regulator of the ER stress response and a pro-survival factor3. XBP-1 splicing assays confirmed the induction of ER stress upon glucose starvation, with elevated levels of XBP-1 mRNA in βIII-tubulin shRNA cells compared with controls. Furthermore, knockdown of βIII-tubulin altered autophagic flux under basal conditions, and this was further exacerbated by glucose starvation. These results strongly indicate that βIII-tubulin knockdown enhances induction of the ER stress response and autophagy in hypoglycemic conditions. CONCLUSION: This study demonstrates for the first time that aberrant βIII-tubulin expression in NSCLC promotes cell survival in hypoglycaemic conditions and modulates the ER stress response and autophagy engaged during glucose starvation. These findings provide new mechanistic insight into the poor prognosis associated with high βIII-tubulin levels in NSCLC. 1 Kavallaris. Nature Rev Cancer, 10:194-204, 2010 2McCarroll et al., Cancer Res 70 :4995-5003, 2010 3Lee, Cancer Res, 67 :3496-3499, 2007 Citation Format: Amelia Parker, Joshua A. McCarroll, Maria Kavallaris. βIII-Tubulin and cell survival: Novel role in endoplasmic reticulum stress and autophagy in non-small cell lung cancer. [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 326. doi:10.1158/1538-7445.AM2014-326

Abstract 4788: MiR-509 inhibits human leukemia cell growth

Abstract Even though the majority of children with precursor B-cell acute lymphoblastic leukemia (B-ALL) can be cured, ALL is still the most frequent cause of cancer-related deaths and causes significant treatment-related toxicity in pediatrics. Thus, there is a need for more effective and less toxic therapies for B-ALL. Specific microRNAs (miRs) may be useful as therapeutics, or to identify downstream therapeutic targets, since miRs regulate essentially all cellular processes. We identified miR-509 via a human genome-wide screen (Cheng WC et al. BioTechniques 2013;54:77. PMC3671589) for miRs that inhibit growth of the NALM6 human B-ALL cell line (harboring the ETV6-PDGFRB fusion gene). MiR microarray expression analysis revealed low endogenous levels of miR-509 in multiple acute leukemia cell lines, CD34+ hematopoietic stem-progenitor cells from normal human donors, and normal B and T lymphocytes. Therefore, enforced expression of miR-509 was used for our mechanistic studies in leukemia cells. Since NALM6 cannot be easily transfected, the precursor miR-509 sequence was cloned downstream of green fluorescence protein (GFP) into a lentiviral vector construct. Enforced lentivector-mediated miR-509 expression was confirmed using RT-qPCR. In a confirmatory growth competition assay measuring the percent GFP-expressing cells by flow cytometry, NALM6 cells transduced with lentiviral miR-509 were progressively outnumbered by mock-transduced cells over five weeks. MiR-509's growth inhibitory effect in NALM6 was further confirmed by orthogonal assays employing trypan blue dye exclusion and alamarBlue fluorescence. MiR-509 similarly inhibited growth of another B-ALL cell line, RCH-ACV (harboring the TCF3-PBX1 fusion gene). In addition, NOD-SCID IL2rg-/- (NSG) mice transplanted miR-509-transduced NALM6 cells had increased survival as compared to empty vector. In order to elucidate the cellular mechanism by which miR-509 exerts its growth inhibitory effects, we performed cell cycle analysis using BrdU/7-AAD staining. MiR-509-transduced NALM6 cells had reduced numbers of S-phase cells, as compared to empty vector-transduced cells. We also observed an increase in apoptotic cell death in miR-509-transduced NALM6 cells, as compared to empty vector-transduced cells. To identify potential targets of miR-509, we used TargetScan to predict targets. We then focused on mRNAs that were expressed in NALM6 cells, as determined by gene expression data in the Cancer Cell Line Encyclopedia. RAB5C was reduced in miR-509 transduced cells as evidenced by qPCR and western blot; luciferase assays indicate that RAB5C is a direct miR-509-3p target. Knockdown of RAB5C by shRNA lentiviral constructs recapitulated the growth inhibitory phenotype induced by miR-509 overexpression in NALM6 cells. In summary, miR-509 suppresses B-ALL cell growth by inhibiting cell proliferation and inducing apoptosis, and miR-509 and RAB5C may be new therapeutic targets for B-ALL. Citation Format: Yee Sun Tan, Wen-Chih Cheng, Curt I. Civin. MiR-509 inhibits human leukemia cell growth. [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 4788. doi:10.1158/1538-7445.AM2014-4788

Abstract 1237: Mechanisms of Vernonia amygdalina-induced apoptosis of human breast adenocarcinoma (MCF-7) cells

Abstract Breast cancer (BC) is the leading cause of death of women between 40 and 55 years of age and is the second overall cause of death of women. Fortunately, the mortality rate from BC has decreased in recent years due to an increased emphasis on early detection and more effective treatments. Despite early detection and conventional methods of treatment, about 7% of women diagnosed with BC still die every year. Therefore, novel therapeutic agents are needed to improve the clinical outcome of this disease. The development of new drugs from natural products is considered important. Previous studies from our laboratory show that a novel natural product, extracts of Vernonia amygdalina (VA) leaf exerts DNA-damaging anticancer activities against BC. Therefore, the central goal of this research was to determine the therapeutic mechanisms of VA leaf extracts in breast cancer cells. To achieve this goal, cell viability, live and death cells were determined by the means of the MTT assay, trypan blue test, and propidium iodine assay, respectively. Cell apoptosis was measured by flow cytometry analysis of phosphatidylserine externalization (Annexin V assay) and caspase 3 activity, and by DNA laddering assay. Data obtained from the MTT assay indicated that VA significantly reduced the viability of MCF-7 cells a dose-dependent response. On one hand, the Trypan blue dye exclusion test demonstrated the integrity of the membrane of untreated cells in culture. On the other hand, the trypan blue dye exclusion test demonstrated a loss of viability in VA-treated cells due to membrane damage. The result of the propidium iodine demonstrated a significant (p&amp;lt;0.05) increase of necrotic cell death in VA-treated cells, indicative of membrane rupture by VA. Flow cytometry data showed a strong dose-response relationship between VA exposure and Annexin-V positive MCF-7 cells. These results were confirmed by data of DNA laddering assay showing a clear evidence of nucleosomal DNA fragmentation in VA-treated cells. A statistically significant was recorded with regard to caspase 3 activity in MCF-7 cells. Taken together, our research demonstrated that VA represents an apoptosis-inducing agent and its apoptotic mechanisms involve phosphatidylserine externalization, activation of p53 tumor suppressor gene and caspase-3, and nucleosomal DNA fragmentation. Citation Format: Clement G. Yedjou, Paul Tchounwou. Mechanisms of Vernonia amygdalina-induced apoptosis of human breast adenocarcinoma (MCF-7) 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 1237. doi:10.1158/1538-7445.AM2014-1237

Abstract 2152: Akt/survivin pathway inhibition synergizes the apoptotic cell death induced by A-santalol in prostate cancer cells

Abstract α-santalol, a major component of sandalwood oil inhibits growth of cultured prostate cancer cells in vitro by causing apoptosis. The present study was undertaken to investigate the mechanistic details associated with the induction of apoptosis by α-santalol in cultured prostate cancer cells (LNCaP and PC-3). Expressions of major proteins studied in the present investigation were determined using standard Western blot protocol and analyzed by LICOR-Odyssey infra-red scanner. Activity of survivin was confirmed employing survivin ELISA kit. The cell viability was determined by trypan blue dye exclusion assay and caspase-3 activity was confirmed using caspase-3 (active) ELISA kit. Treatment of prostate cancer cells with α-santalol (20, 40 µM) resulted in the down regulation of survivin, XIAP and p-AKT (s-473) levels. Furthermore, α-santalol significantly reduced the activity of survivin as evidenced by survivin ELISA assay. Inhibition of PI3K/Akt by pharmacological inhibitor LY294002 synergized the apoptotic cell death induced by α-santalol as determined by cell viability, active caspase-3 activity and expression of cleaved PARP, caspase-3 levels. In conclusion, the present study reveals that α-santalol downregulates activity and expression of survivin and that inhibition of PI3K/Akt pathway synergize the apoptotic cell death by downregulating survivin. This study was supported by Mentoring Task Force, Type I grant by Wilkes University and by Translational Cancer Research Center funded by South Dakota Governor's Office of Economic Development. Citation Format: Ajay Bommareddy, Lauren Lockus, Jonathan Seward, William Eggelston, Stacy Prelewicz, Andrea Antal, Sarah Fillman, Christian Castro, Adam L. VanWert, Sreevidya Santha, Chandradhar Dwivedi. Akt/survivin pathway inhibition synergizes the apoptotic cell death induced by A-santalol 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 2152. doi:10.1158/1538-7445.AM2014-2152

Abstract 345: Sensitivity of human leukemic sup-t1 cells to chemotherapeutic agents

Abstract Background: Lymphoid cell apoptosis plays an important role in the development and functioning of the immune system. The ability of chemotherapeutic agents such as antimetabolites, topoisomerase poisons, alkylating agent mimics, anti-tumor antibiotics, and glucocorticoids to target the apoptotic pathway is instrumental in their potency as anti-leukemic agents. Resistance to chemotherapeutic agents is a hindrance to disease-free survival. An understanding of the genetic basis for apoptosis induction is essential to the development of more effective chemotherapeutic regimens. Studies in our laboratory, and by other investigators, have suggested that induction of a proapoptotic Bcl-2 family member, Bim, facilitates drug-induced apoptosis of T-lymphocytic leukemia cells in culture. We have identified E4BP4 and Bcl11b as transcriptional regulators of Bim expression. Both E4BP4 and Bcl11b are vital for normal immune cell development and are known to have reciprocal effects on T-cell differentiation and survival. E4BP4 is required for maturation of Induced T-cell derived Natural Killer cells (ITNK) while Bcl11b expression is required for maintenance of T-cell identity. In separate experiments, Bcl11b repression and E4BP4 upregulation have been associated with T-cell apoptosis. In the studies presented here, we are establishing a relationship between E4BP4 and Bcl11b expression, leukemic cell apoptosis, and sensitivity to chemotherapeutic agents in cell culture models of T-lympboblastic leukemia. Methods: The sensitivity of human Leukemic T-lymphoblast cell line, Sup-T1, to each class of anti-leukemic agent is being evaluated by trypan blue dye exclusion assays and flow cytometric analysis. Drug-induced alterations in E4BP4, Bcl11b, and Bim transcripts were evaluated by RT-qPCR, and the data are being interpreted via Pfaffl analysis. Results: Sup-T1 cells were resistant to the synthetic glucocorticoid Dexamethasone, and the alkylating agent mimic Cisplatin. Sup-T1 showed moderate sensitivity to the topoisomerase poison Etoposide. Marked sensitivity to the anti-tumor antibiotic Daunorubicin and the antimetabolite 5-fluorouracil was indicated. E4BP4 and Bim transcript levels seem to be induced by Daunorubicin and 5-fluorouracil, when compared to the reference gene β-actin. Bcl11b regulation is being investigated currently and all results are being tested against a new reference gene, GAPDH. Conclusions: Our data support our hypothesis of a correlation between sensitivity to chemotherapeutic agents and upregulation of E4BP4 and Bim in Sup-T1 cells. Analysis of Bcl11b expression data is ongoing. Acknowledgements: This project is funded by NIH grant (SC3 GM 081099) awarded to RDM. Citation Format: Andrew D. Gisis, Rheem D. Medh. Sensitivity of human leukemic sup-t1 cells to chemotherapeutic agents. [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 345. doi:10.1158/1538-7445.AM2014-345

Synthesis and evaluation of novel benzophenone-thiazole derivatives as potent VEGF-A inhibitors

A series of 2-(4-benzoyl-phenoxy)-N-(4-phenyl-thiazol-2-yl)-acetamides (10a–n) were synthesized by multistep reaction sequence and all the compounds were well characterized for structural elucidation. The in vitro cytotoxicity of compounds 10a–n was evaluated against EAC and DLA cell lines using trypan blue dye exclusion method. Further MTT assay and LDH release assay, followed by in vivo studies on murine model were also evaluated. The compound 10h with a methyl and fluoro groups at benzophenone moiety and methoxy group at phenyl ring was in a leading position to exhibit the promising antiproliferative effect through translational VEGF-A inhibition.

Tegillarca granosa extract Haishengsu induces apoptosis in human hepatocellular carcinoma cell line BEL-7402 via Fas-signaling pathways.

This study was designed to investigate the apoptosis-inducing properties of Tegillarca granosa extract Haishengsu (HSS) in human hepatocellular carcinoma cell line BEL-7402. Proliferation inhibition of the human hepatocellular carcinoma BEL-7402 cells was determined by the MTT assay, and the cell viability was determined by trypan blue dye exclusion assay. Apoptosis of BEL-7402 cells was demonstrated by fluorescence microscope with flow cytometry with Annexin V-FITC/PI double staining and Hoechst 33258 staining. Western blot analysis and RT-PCR were used to determine the expression levels of Fas. Expressions of caspase-8 and caspase-3 were examined by caspase activity assay and western blot analysis. HSS inhibited the proliferation of human hepatocellular carcinoma BEL-7402 cells in a dose- and time-dependent manner. Our results showed HSS had positive effect on apoptosis through flow cytometry assay and fluorescence microscope. The expressions of Fas protein and mRNA were up-regulated following the treatment. Caspase-8 and caspase-3 were activated in the cells cultured with HSS. In conclusion, HSS induced apoptosis of human hepatocellular carcinoma BEL-7402 cells. The apoptosis was associated with the up-regulation of Fas and the activations of caspase-8 and caspase-3.

Leishmanicidal compounds and potent PPARγ activators from Renealmia thyrsoidea (Ruiz & Pav.) Poepp. & Endl.

Ethnopharmacological relevanceLeaves and rhizomes of Renealmia thyrsoidea (Ruiz & Pav.) Poepp. & Endl. traditionally used in the Yanesha pharmacopoeia to treat skin infections such as leishmaniasis ulcers, or to reduce fever were chemically investigated to identify leishmanicidal compounds, as well as PPARγ activators. MethodsCompounds were isolated through a bioassay-guided fractionation and their structures were determined via detailed spectral analysis. The viability of Leishmania amazonensis axenic amastigotes was assessed by the reduction of tetrazolium salt (MTT), the cytotoxicity on macrophage was evaluated using trypan blue dye exclusion method, while the percentage of infected macrophages was determined microscopically in the intracellular macrophage-infected assay. The CD36, mannose receptor (MR) and dectin-1 mRNA expression on human monocytes-derived macrophages was evaluated by quantitative real-time PCR. ResultsSix sesquiterpenes (1–6), one dihydrobenzofuranone (7) and four flavonoids (8–11) were isolated from the leaves. Alongside, two flavonoids (12–13) and five diarylheptanoids (14–18) were identified in the rhizomes. Leishmanicidal activity against Leishmania amazonensis axenic amastigotes was evaluated for all compounds. Compounds 6, 7, and 11, isolated from the leaves, showed to be the most active derivatives. Diarylheptanoids 14–18 were also screened for their ability to activate PPARγ nuclear receptor in macrophages. Compounds 17 and 18 bearing a Michael acceptor moiety strongly increased the expression of PPARγ target genes such as CD36, Dectin-1 and mannose receptor (MR), thus revealing interesting immunomodulatory properties. ConclusionsPhytochemical investigation of Renealmia thyrsoidea has led to the isolation of leishmanicidal compounds from the leaves and potent PPARγ activators from the rhizomes. These results are in agreement with the traditional uses of the different parts of Renealmia thyrsoidea.