Significant Induction Of Cell Death Research Articles

The Apoptotic Potential Activity of Terminalia Brownii Extracts against Various Cancer Cell Lines.

The present study aimed to investigate the potential inhibitory activities of various Terminalia brownie extracts against the proliferation of different cancer cell lines. Fractionation was carried out through the process of liquid-liquid extraction produced four fraction types using polarity-enhancing solvents such as, n-hexane, chloroform, ethyl acetate, and water. All extracts have been tested for their cytotoxicity against cancer cell lines using SRB assay and their inhibitory activity on cell cycle phases using flow cytometry. The chloroform extract exhibited a cytotoxic effect against all three types of cancer cell lines and especially the HepG2 cells at 100-µg concentration. Moreover, the most prominent plant extracts of chloroform, acetyl acetate, and hexane showed significant induction of cell death during the S phase cell cycle. The findings of this study suggest that compounds in the Terminalia brownie plant used in traditional Saudi medicine may have the potential to suppress the growth of cancer cell lines.

Environmental Impact of Pharmaceutical Pollutants: Synergistic Toxicity of Ivermectin and Cypermethrin

Veterinary antiparasitic pharmaceuticals as well as pesticides have been detected in surface waters, and they may cause several toxic effects in this environmental compartment. In the present study, we evaluated the toxicity after exposure of different concentration of ivermectin (IVM; 50, 100, and 200 μg L−1) and cypermethrin (CYP; 5, 10, and 25 μg L−1) and the combination of these two compounds at non-toxic concentration (IVM 100 + CYP 5 μg L−1) in zebrafish embryos. Combination of IVM at 100 μg L−1 with CYP at 5 μg L−1 exposure induced hatching delay and malformations at 96 hpf in zebrafish larvae as well as significant induction of cell death in zebrafish larvae. At the same time, the two single concentrations of IVM and CYP did not show a toxic effect on zebrafish development. In conclusion, our study suggests that IVM and CYP show a synergistic effect at common, ineffective concentrations, promoting malformation and cell death in fish development.

Cerium Oxide Nanoparticles: Synthesis and Characterization for Biosafe Applications

Due to its excellent physicochemical properties, cerium oxide (CeO2) has attracted much attention in recent years. CeO2 nanomaterials (nanoceria) are widely being used, which has resulted in them getting released to the environment, and exposure to humans (mostly via inhalation) is a major concern. In the present study, CeO2 nanoparticles were synthesized by hydroxide-mediated method and were further characterized by Scanning Electron Microscopy (SEM), Field Emission Scanning Electron Microscopy (FESEM), Energy Dispersive X-ray Spectroscopy (EDX), Fourier Transform Infrared Spectroscopy (FTIR), and X-ray Diffraction Spectroscopy (XRD). Human lung epithelial (Beas-2B) cells were used to assess the cytotoxicity and biocompatibility activity of CeO2 nanoparticles. 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) and Live/Dead assays were performed to determine the cytotoxicity and biocompatibility of CeO2 nanoparticles. Generation of reactive oxygen species (ROS) by cerium oxide nanoparticles was assessed by ROS assay. MTT assay and Live/Dead assays showed no significant induction of cell death even at higher concentrations (100 μg per 100 μL) upon exposure to Beas-2B cells. ROS assay revealed that CeO2 nanoparticles did not induce ROS that contribute to the oxidative stress and inflammation leading to various disease conditions. Thus, CeO2 nanoparticles could be used in various applications including biosensors, cancer therapy, catalytic converters, sunscreen, and drug delivery.

Fucoxanthin Holds Potential to Become a Drug Adjuvant in Breast Cancer Treatment: Evidence from 2D and 3D Cell Cultures.

Fucoxanthin (Fx) is a carotenoid derived from marine organisms that exhibits anticancer activities. However, its role as a potential drug adjuvant in breast cancer (BC) treatment is still poorly explored. Firstly, this study investigated the cytotoxic effects of Fx alone and combined with doxorubicin (Dox) and cisplatin (Cis) on a panel of 2D-cultured BC cell lines (MCF7, SKBR3 and MDA-MB-231) and one non-tumoral cell line (MCF12A). Fucoxanthin induced cytotoxicity against all the cell lines and potentiated Dox cytotoxic effects towards the SKBR3 and MDA-MB-231 cells. The combination triggering the highest cytotoxicity (Fx 10 µM + Dox 1 µM in MDA-MB-231) additionally showed significant induction of cell death and genotoxic effects, relative to control. In sequence, the same combination was tested on 3D cultures using a multi-endpoint approach involving bioactivity assays and microscopy techniques. Similar to 2D cultures, the combination of Fx and Dox showed higher cytotoxic effects on 3D cultures compared to the isolated compounds. Furthermore, this combination increased the number of apoptotic cells, decreased cell proliferation, and caused structural and ultrastructural damages on the 3D models. Overall, our findings suggest Fx has potential to become an adjuvant for Dox chemotherapy regimens in BC treatment.

Evaluation of Cellulose Nanoparticles Synthesized by Acid Hydrolysis for Safe Applications

In the present study, cellulose nanoparticles were synthesized from waste cotton by acid hydrolysis method. Nanoparticles were obtained by the hydrolysis of pretreated cotton fibers with an acid mixture containing water, hydrochloric acid (HCl) and sulphuric acid (H2SO4). Characterization and biocompatibility studies of obtained cellulose nanoparticles were performed successfully. The morphological studies of cellulose nanoparticles were conducted by Field Emission Scanning Electron Microscopy (FESEM) and Transmission Electron Microscopy (TEM). Both showed that the cellulose nanoparticles synthesized from waste cotton measured approximately 100 - 200nm in diameter. Energy Dispersive X-ray Spectroscopic spectrum showed the elemental composition of the cellulose nanoparticles containing carbon and oxygen. Fourier Transform Infrared (FT-IR) spectroscopy spectrum revealed the presence of several characteristic peaks of cellulose nanoparticles. Human lung epithelial (Beas-2B) cells were used to assess the cytotoxicity and biocompatibility activity of cellulose nanoparticles. Cytotoxicity of cellulose nanoparticles was determined by 3-(4,5- Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay. Live/Dead viability assay was carried out to assess the biocompatibility of cellulose nanoparticles. Generation of Reactive Oxygen Species in Beas-2B cells incubated with cellulose nanoparticles was determined by Reactive Oxygen Species (ROS) assay. MTT and Live/Dead assays showed no significant induction of cell death even at higher concentrations (100 μg) upon exposure to Beas- 2B cells. ROS assay revealed that cellulose nanoparticles did not induce any reactive oxygen species that contribute to the oxidative stress and inflammation leading to various disease conditions. The results revealed that the cellulose nanoparticles have a great potential in a variety of commercial applications

Inhibition of telomerase activity by splice-switching oligonucleotides targeting the mRNA of the telomerase catalytic subunit affects proliferation of human CD4+ T lymphocytes

Telomerase activity is regulated at the mRNA level by alternative splicing (AS) of its catalytic subunit hTERT. The aim of this study was to define the ability of splice-switching oligonucleotides (SSOs) that pair with hTERT pre-mRNA to induce AS and inhibit telomerase activity in human CD4+ T lymphocytes. SSOs that blocked the binding of a single splicing regulatory protein, SRp20 or SRp40, to its site within intron 8 of hTERT pre-mRNA demonstrated rather moderate capacities to induce AS and inhibit telomerase. However, a SSO that blocked the interaction of both SRp20 and SRp40 proteins with pre-mRNA was the most active. Cultivation of lymphocytes with spliced hTERT and inhibited telomerase resulted in the reduction of proliferative activity without significant induction of cell death. These results should facilitate further investigation of telomerase activity regulation, and antitelomerase SSOs could become promising agents for antiproliferative cell therapy.

Effects of FOXM1 inhibition and ionizing radiation on melanoma cells.

Metastatic melanoma can be highly refractory to conventional radiotherapy and chemotherapy but combinatorial-targeted therapeutics are showing greater promise on improving treatment efficacy. Previous studies have shown that knockdown of Forkhead box M1 (FOXM1) can sensitize various tumor types to radiation-induced cell death. The effect of combining radiation with a small molecule FOXM1 inhibitor, Siomycin A, on growth, death and migration of a metastatic melanoma cell line (SK-MEL-28) that overexpresses this pleiotropic cell cycle regulator was investigated. Siomycin A (SIOA) was found to be a strong inducer of apoptosis, and inhibitor of proliferation and migration in a scratch wound assay in this cell line. Induction of apoptosis occurred at concentrations >1 µM in association with reductions in the constitutive FOXM1 and anti-apoptotic B-cell lymphoma 2 protein levels found in these cells. Single doses of ionizing radiation (0-40 Gy) delivered by linear accelerator caused inhibition of growth and migration without significant induction of cell death. Pretreatment with SIOA did not increase the sensitivity of this melanoma cell line to radiation as observed in other tumor types. These data confirm that as a single agent, SIOA is an effective inducer of cell death and inhibitor of migration in metastatic melanoma cells expressing constitutive FOXM1. In combination with radiation, SIOA pre-treatment, however, may not be of added benefit.

Enzymatic Synthesis, Characterization and Biocompatibility Studies of Cellulose Nanoparticles from Cotton Fibers

Interest in cellulose nanoparticles has been increasing exponentially in the past few decades due to its unique characteristics such as reinforcement properties, high tensile strength, and excellent thermal and electrical properties. Cellulose nanoparticles were produced by an enzymatic method including hydrolysis of cotton fibers by cellulase enzyme and sonication process. Further, cellulose nanoparticles were characterized to determine the morphology and purity of the material. Characterization of cellulose nanoparticles was performed by Scanning Electron Microscope (SEM) with Energy Dispersive X-Ray Spectroscopy (EDX) and Fourier Transform Infrared Spectroscopy (FT-IR). Biocompatibility studies of cellulose nanoparticles were carried out by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay and Live/Dead viability assay. Using SEM, the average size of cellulose nanoparticles was found to be around 100-125nm and the particles were spherical in shape. FTIR spectrum showed the formation of cellulose nanoparticles from cotton fibers without any presence of impurities. MTT assay and Live/Dead viability assay showed no significant induction of cell death even at higher concentrations (100 μg) upon exposure to Rat Lung Epithelial cells. The results revealed that the synthesized cellulose nanoparticles could be used in wide range of emerging applications in the development of new energy storage devices, enzyme immobilization, antimicrobial and medical materials, green catalysis, bio-sensing and controlled drug delivery.

Cytotoxic and apoptotic activities of extract of Amaranthus spinosus L. in Allium cepa and human erythrocytes.

The present study examined the apoptosis inducing effects of Amaranthus spinosus L. aqueous extract in Allium cepa root meristematic cells and human erythrocytes. Cytogenetic assay revealed many apoptosis inducing cytogenetic aberrations viz., cytoplasmic breakage, cytoplasmic disintegration, cytoplasmic shrinkage, receding of cytoplasm, cytoplasmic vacuolation, enucleated cell, ghost cell, nuclear vacuolation, nuclear fragmentation and nuclear disintegration. A remarkable modification of red blood cell surface morphology was observed in the result of RBC assay. The treated RBCs show membrane blebbing and shrinkage, features typical for apoptosis in nucleated cells. Significant induction of cell death was observed in treated Allium root tip cells after Evans blue staining, disclosing the membrane damage potential of the plant extract. TTC assay results in reduced mitochondrial/metabolic activity in Allium root tip cells after treatment, designating the adverse effect of plant extract on mitochondrial respiratory chain. These results confirm the apoptosis inducing potential of A. spinosus extract. Confirming the present results by further in vitro studies, it can be effectively targeted against cell proliferation during cancer treatment by inducing apoptosis. Thus from the present investigation it can be concluded that the aqueous extract of A. spinosus exhibited apoptosis induction and cytotoxic activities.

Abstract P5-03-10: CYC065, a novel CDK2/9 inhibitor: Molecular basis for clinical development in basal-like triple-negative breast cancer

Abstract CYC065 is a novel CDK inhibitor, which inhibits CDK2 and 9 with IC50 values of 5 and 26 nM, respectively. Following completion of IND-enabling studies, CYC065 has been cleared by FDA for first-in-human Phase 1 clinical trials. Triple-negative breast cancers (TNBC), particularly the basal subtype, often exhibit aggressive characteristics. Despite good initial responses to chemotherapy, patients experience early relapse and diminished 5 year survival. Molecular features of basal-like TNBC include amplification or overexpression of cyclin E and MYC, suggesting potential utility for a CDK2/9 inhibitor such as CYC065. CYC065 is effective in cyclin E-overexpressing tumors, such as uterine serous carcinoma1 and trastuzumab-resistant Her2+ breast cancer2. Moreover, CDK inhibition has also been reported to be synthetic lethal with overexpressed MYC3. This led us to assess the potency and mechanism of action of CYC065 in basal-like TNBC models to evaluate the potential for CYC065 development in this indication. In vitro cell-based experiments support twice weekly pulse dosing using submicromolar concentrations of CYC065 to achieve maximum impact on cell growth in the majority of breast cancer cell lines tested. Preclinical toxicology data indicate that such levels and durations of exposure are achievable and well tolerated. As a single agent, CYC065 treatment in breast cancer cells resulted in inhibition of RNA-Pol II phosphorylation, down-regulation of Mcl-1, up-regulation of p53 and rapid induction of apoptosis. The impact of CYC065 on CDK2 targets, cyclin E and MYC was also explored. Interestingly immortalized cell lines obtained from non-malignant tissue displayed similar effects on RNA Pol II, Mcl-1, and p53 but did not undergo apoptosis and consequently exhibited relative resistance to CYC065, indicative of a potential therapeutic window. Cell cycle analysis demonstrated that CYC065 treatment induced an increase in G1 population with no significant induction of cell death in non-malignant derived cell lines, compared to cancer cell lines, in which there was significant induction of cell death. CDKs have a role in DNA repair which can be exploited to enhance the effectiveness of DNA damaging agents. Seliciclib, an oral, first generation CDK2/9 inhibitor, can be effectively combined with DNA damaging agents, such as the oral nucleoside analogue sapacitabine, or its active metabolite CNDAC. A Phase 1 clinical trial is currently underway to evaluate this combination (NCT00999401). Similarly to seliciclib, we demonstrate that CYC065 is synergistic in combination with CNDAC when given sequentially across multiple breast cancer cell lines. CNDAC-induced double strand breaks persisted for longer when cells were subsequently treated with CYC065, supporting the conclusion that these CDK inhibitors suppress DNA double-strand break repair capacity, which may contribute to the observed synergy. Taken together the data establish CYC065 as a promising anti-cancer agent in basal-like TNBC, with the potential to be combined effectively in this indication with DNA damaging agents. 1. Cocco, E. et al. AACR, 2015, Abstract 3103. 2. Scaltriti, M. et al. Proc Natl Acad Sci USA. 201, 108, 3761-6. 3. Horiuchi, D. et al. J Exp Med. 2012, 209, 679-96. Citation Format: Blake DG, MacKay C, Frame S, Zheleva D. CYC065, a novel CDK2/9 inhibitor: Molecular basis for clinical development in basal-like triple-negative breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-03-10.

SK-N-SH 신경세포내 항산화 효과와 p38 인산화 억제에 의한 곤드레, 누룩치 그리고 산마늘의 신경 보호 효과

Oxidative stress is one of the key mechanisms involved in neuronal damage. Neuroprotective effects and underlying mechanisms of action of several wild vegetables, Cirsium setidens (CS), Pleurospermum kamtschaticumin (PK), and Allium victorials (AV), against oxidative stress induced by hydrogen peroxide in SK-N-SH cells were investigated. CS and AV up to 400 μg/mL showed no detectable effects on cell viability of human SK-N-SH neuroblastoma cells compared with control. Incubation of SK-N-SH cells with hydrogen peroxide resulted in significant induction of cell death and reaction oxygen species (ROS) production, whereas treatment of cells with CS and AV significantly reduced cell death and ROS production, respectively. Among the wild vegetables tested, CS and PK showed more effective DPPH radical scavenging activity than AV, whereas PK showed strong cytotoxicity in SK-N-SH cells compared with the control. CS showed much higher inhibitory effects on cell death and ROS generation against oxidative stress than AV. Thus, CS was selected for subsequent experiments. Ethyl acetate (EA), hexane, butanol, aqueous, and chloroform extracts from CS significantly inhibited cell death and ROS generation in SK-N-SH cells induced by oxidative stress. EA extract from CS (CS-EA) showed the highest DPPH radical-scavenging activity, intracellular ROS-scavenging activity, and neuroprotective effects. CS-EA attenuated apoptosis signal-regulating p38 activation by inhibiting phosphorylation. The findings suggest that CS-EA protects neuronal cells through antioxidant activity and inhibition of phosphorylation of p38 in brain neural cells.

RITA can induce cell death in p53-defective cells independently of p53 function via activation of JNK/SAPK and p38.

Significant advances have been made in the development of small molecules blocking the p53/MDM2 interaction. The Mdm2 inhibitor Nutlin-3 is restricted to tumors carrying wtp53. In contrast, RITA, a compound that binds p53, has recently been shown also to restore transcriptional functions of mtp53. As more than 50% of solid tumors carry p53 mutations, RITA promises to be a more effective therapeutic strategy than Nutlin-3. We investigated effects of RITA on apoptosis, cell cycle and induction of 45 p53 target genes in a panel of 14 cell lines from different tumor entities with different p53 status as well as primary lymphocytes and fibroblasts. Nine cell strains expressed wtp53, four harbored mtp53, and three were characterized by the loss of p53 protein. A significant induction of cell death upon RITA was observed in 7 of 16 cell lines. The nonmalignant cells in our panel were substantially less sensitive. We found that in contrast to Nultin-3, RITA is capable to induce cell death not only in tumor cells harboring wtp53 and mtp53 but also in p53-null cells. Importantly, whereas p53 has a central role for RITA-mediated effects in wtp53 cells, neither p53 nor p63 or p73 were essential for the RITA response in mtp53 or p53-null cells in our panel demonstrating that besides the known p53-dependent action of RITA in wtp53 cells, RITA can induce cell death also independently of p53 in cells harboring defective p53. We identified an important role of both p38 and JNK/SAPK for sensitivity to RITA in these cells leading to a typical caspase- and BAX/BAK-dependent mitochondrial apoptosis. In conclusion, our data demonstrate that RITA can induce apoptosis through p38 and JNK/SAPK not only in tumor cells harboring wtp53 and mtp53 but also in p53-null cells, making RITA an interesting tumor-selective drug.

Microglial Cell Activation Increases Saturated and Decreases Monounsaturated Fatty Acid Content, but Both Lipid Species are Proinflammatory

Neuroinflammation is a component of age-related neurodegenerative diseases and cognitive decline. Saturated (SFA) and monounsaturated (MUFA) fatty acids are bioactive molecules that may play different extrinsic and intrinsic roles in neuroinflammation, serving as exogenous ligands for cellular receptors, or endogenous components of cell structural, energetic and signaling pathways. We determined the fatty acyl profile of BV2 microglial cells before and after acute activation with lipopolysaccharide (LPS). We also investigated the effect of SFA and MUFA pretreatment on the production of an invasive, neurotoxic phenotype in BV2 cells. Acute activation of BV2 microglia resulted in an increase in the relative content of SFA (12:0, 16:0, 18:0, 20:0, 22:0, and 24:0 increased significantly), and a relative decrease in the content of MUFA (16:1n7, 18:1n7, 18:1n9, 20:1n9, 24:1n9 decreased significantly). In agreement, the major stearoyl-CoA desaturase (SCD) isoform in BV2 cells, SCD2, was significantly down-regulated by LPS. We next treated cells with SFA (16:0 or 18:0) or MUFA (16:1n7 or 18:1n9), and found that levels of secreted IL6 were increased, as was secreted MMP9-mediated proteolytic activity. To test the functional significance, we treated SH-SY5Y neuronal cells with conditioned medium from BV2 cells pretreated with fatty acids, and found a small but significant induction of cell death. Our findings suggest differential intrinsic roles for SFA and MUFA in activated microglial cells, but similar extrinsic roles for these fatty acid species in inducing activation. Expansion of SFA is important during microglial cell activation, but either supplemental SFA or MUFA may contribute to chronic low-grade neuroinflammation.

PI3K/mTOR Inhibition Upregulates NOTCH-MYC Signalling Leading to an Impaired Cytotoxic Response

Abstract 1358The PI3K/mTOR and NOTCH pathways are promising therapeutic targets for the treatment of T-ALL. Hyperactivation of the PI3K/mTOR pathway occurs frequently, predominantly due to loss of PTEN function through deletion, mutation, microRNA induced downregulation or post-translational modification. NOTCH signalling is aberrantly activated in the majority of patients, most commonly due to mutation of Notch-1. Activation of NOTCH signalling can also positively regulate mTOR activity and increase PI3K/Akt signalling via downregulation of PTEN expression.We examined the effects of PI3K/mTOR blockade, using the dual inhibitor PI-103, on the proliferation and survival of T-ALL cell lines with various combinations of NOTCH and PTEN abnormalities. There was marked reduction in the proliferation of all T-ALL cell lines tested, regardless of their PTEN status or level of activated Akt. However, using Annexin-V/PI staining, we observed significant induction of cell death (<50% survival) in only 3/15 cell lines.Blockade of NOTCH signalling, using a gamma secretase inhibitor (GSI), had no effect on cell survival and only a modest effect on cell proliferation: only 5/13 NOTCH deregulated cell lines, all of which expressed wild-type PTEN, showed a clear reduction in cell number. By contrast only 1 of the 8 GSI-resistant cell lines expressed wild-type PTEN.We tested the effect of combined blockade of PI3K/mTOR and NOTCH pathways using PI-103 + GSI to determine the extent of any non-overlapping effects. In NOTCH-mutant/PTEN-WT cells, combined blockade led to a reduction in cell size and number with a more rapid and marked increase in cell cycle arrest and reduced levels of Cdk4 and Cyclin D3, than achieved with either agent alone. Further, 8/13 cell lines with deregulated Notch showed a significant reduction in cell survival with PI-103+GSI compared with PI-103 alone. In 5/6 NOTCH-mutant/PTEN-WT cell lines survival fell to ≤50% below control, with rapid commitment to cell death (48–72 hours). This mutational context represents the majority of primary T-ALL samples at presentation - confirmation of the enhanced effect of dual PI3K/mTOR and NOTCH blockade was obtained in primary T-ALLs cultured in suspension or on stromal support. Utilizing selective inhibitors of the PI3K (PIK90) or mTOR (rapamycin) enzymes in combination with GSI we found that blockade of both enzymes was required to achieve maximal levels of cell death.c-MYC is an important oncogene in T-ALL - it is a direct transcriptional target of Notch signaling and protein stability is modulated via the PI3K/Akt/GSK3 module. Therefore, we examined the effects of PI3K/mTOR and NOTCH blockade on levels of nuclear c-MYC in cells sensitive to inhibition of both pathways. c-MYC was downregulated by GSI but, surprisingly, increased after 48h of PI-103 treatment. This followed increased levels of nuclear Notch intracellular domain and could be abolished by the addition of GSI, indicating a NOTCH-dependent mechanism. Gene expression microarray analysis confirmed global upregulation of NOTCH signalling in PI-103 treated cells and the suppression of this effect by addition of GSI. These findings were confirmed for Notch target genes (Deltex-1, c-MYC, Hes-1, CD21 and GIMAP5) by qPCR and flow cytometry (CD21).Upregulation of Notch-MYC proliferation and survival signals could explain why T-ALL cells survive after PI3K/mTOR blockade. We found that maintenance of c-MYC levels by retroviral expression counteracted the effects of PI3K/mTOR/NOTCH blockade and comparable levels of cell death, to those seen with PI-103 plus GSI, were seen in cells treated with PI-103 + the c-MYC inhibitor, 10058-F4.Our data show that targeting PI3K/mTOR can upregulate NOTCH-MYC activity in T-ALL cells with aberrant NOTCH signalling. These finding have implications for the use of PI3K inhibitors for the treatment of T-ALL, and other malignancies with activated NOTCH signaling, and provide a rational basis for the use of drug combinations that target both pathways. Disclosures:No relevant conflicts of interest to declare.

Cytotoxic effects of polycarbonate-based orthodontic brackets by activation of mitochondrial apoptotic mechanisms

ObjectivesThe aim of the study was to evaluate the biological effects of water eluents from polycarbonate based esthetic orthodontic brackets. MethodsThe composite polycarbonate brackets tested were Silkon Plus (SL, fiber-glass-reinforced), Elan ME (EL, ceramic particle-reinforced) and Elegance (EG, fiber-glass-reinforced). An unfilled polyoxymethylene bracket (Brilliant, BR) was used as control. The brackets’ composition was analyzed by ATR-FTIR spectrometry. The cytotoxicity and estrogenicity of the eluents obtained after 3months storage of the brackets in water (37°C) were investigated in murine fibroblasts (NIH 3T3), breast (MCF-7) and cervical cancer (CCl-2/Hela) cell lines. ResultsSL and EG were based on aromatic-polycarbonate matrix, whereas EL consisted of an aromatic polycarbonate-polyethylene terepthalate copolymer. A significant induction of cell death and a concurrent decrease in cell proliferation was noted in the EG eluent-treated cells. Moreover, EG eluent significantly reduced the levels of the estrogen signaling associated gene pS2, specifically in MCF7 cells, suggesting that cell death induced by this material is associated with downregulation of estrogen signaling pathways. Even though oxidative stress mechanisms were equally activated by all eluents, the EG eluents induced expression of apoptosis inducing factor (AIF) and reduced Bcl-xL protein levels. SignificanceSome polycarbonate-based composite brackets when exposed to water release substances than activate mitochondrial apoptosis.

The role of microRNA deregulation in the pathogenesis of adrenocortical carcinoma

Adrenocortical carcinoma (ACC) is an aggressive tumor showing frequent metastatic spread and poor survival. Although recent genome-wide studies of ACC have contributed to our understanding of the disease, major challenges remain for both diagnostic and prognostic assessments. The aim of this study was to identify specific microRNAs (miRNAs) associated with malignancy and survival of ACC patients. miRNA expression profiles were determined in a series of ACC, adenoma, and normal cortices using microarray. A subset of miRNAs showed distinct expression patterns in the ACC compared with adrenal cortices and adenomas. Among others, miR-483-3p, miR-483-5p, miR-210, and miR-21 were found overexpressed, while miR-195, miR-497, and miR-1974 were underexpressed in ACC. Inhibition of miR-483-3p or miR-483-5p and overexpression of miR-195 or miR-497 reduced cell proliferation in human NCI-H295R ACC cells. In addition, downregulation of miR-483-3p, but not miR-483-5p, and increased expression of miR-195 or miR-497 led to significant induction of cell death. Protein expression of p53 upregulated modulator of apoptosis (PUMA), a potential target of miR-483-3p, was significantly decreased in ACC, and inversely correlated with miR-483-3p expression. In addition, high expression of miR-503, miR-1202, and miR-1275 were found significantly associated with shorter overall survival among patients with ACC (P values: 0.006, 0.005, and 0.042 respectively). In summary, we identified additional miRNAs associated with ACC, elucidated the functional role of four miRNAs in the pathogenesis of ACC cells, demonstrated the potential involvement of the pro-apoptotic factor PUMA (a miR-483-3p target) in adrenocortical tumors, and found novel miRNAs associated with survival in ACC.

Bortezomib Sensitizes Malignant Human Glioma Cells to TRAIL, Mediated by Inhibition of the NF-κB Signaling Pathway

Previous studies have shown that the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has significant apoptosis-inducing activity in some glioma cell lines, although many lines are either moderately or completely resistant, which has limited the therapeutic applicability of this agent. Because our recent studies showed that inhibition of proteasomal function may be independently active as an apoptosis-inducing stimulus in these tumors, we investigated the sensitivity of a panel of glioma cell lines (U87, T98G, U373, A172, LN18, LN229, LNZ308, and LNZ428) to TRAIL alone and in combination with the proteasome inhibitor bortezomib. Analysis of these cell lines revealed marked differences in their sensitivity to these treatments, with two (LNZ308 and U373) of the eight cell lines revealing no significant induction of cell death in response to TRAIL alone. No correlation was found between sensitivity of cells to TRAIL and expression of TRAIL receptors DR4, DR5, and decoy receptor DcR1, caspase 8, apoptosis inhibitory proteins XIAP, survivin, Mcl-1, Bcl-2, Bcl-Xl, and cFLIP. However, TRAIL-resistant cell lines exhibited a high level of basal NF-κB activity. Bortezomib was capable of potentiating TRAIL-induced apoptosis in TRAIL-resistant cells in a caspase-dependent fashion. Bortezomib abolished p65/NF-κB DNA-binding activity, supporting the hypothesis that inhibition of the NF-κB pathway is critical for the enhancement of TRAIL sensitization in glioma cells. Moreover, knockdown of p65/NF-κB by shRNA also enhanced TRAIL-induced apoptosis, indicating that p65/NF-κB may be important in mediating TRAIL sensitivity and the effect of bortezomib in promoting TRAIL sensitization and apoptosis induction.

Anti-apoptotic Effects of Red Ginseng on Oxidative Stress Induced by Hydrogen Peroxide in SK-N-SH Cells

Ginseng (Panax ginseng C.A. Meyer) has been shown to have anti-stress effects in animal studies. However, most studies have only managed to detect altered levels of biomarkers or enzymes in blood or tissue, and the actual molecular mechanisms by which ginseng exerts these effects remain unknown. In this study, the anti-oxidative effect of Korean red ginseng (KRG) was examined in human SK-N-SH neuroblastoma cells. Incubation of SK-N-SH cells with the oxidative stressor hydrogen peroxide resulted in significant induction of cell death. In contrast, pre-treatment of cells with KRG decreased cell death significantly. To elucidate underlying mechanisms by which KRG inhibited cell death, the expression of apoptosis-related proteins was examined by Western blot analysis. KRG pre-treatment decreased the expression of the pro-apoptotic gene caspase-3, whereas it increased expression of the anti-apoptotic gene Bcl-2. Consistent with this, immunoblot analysis showed that pre-treatment of the SK-N-SH cells with KRG inhibited expression of the pro-inflammatory gene cyclooxygenase 2 (COX-2). RT-PCR analysis revealed that the repression of COX-2 expression by KRG pre-treatment occurred at the mRNA level. Taken together, our data indicate that KRG can protect against oxidative stress-induced neuronal cell death by repressing genes that mediate apoptosis and inflammation.

Regulation of IGFBP6 gene and protein is mediated by the inverse expression and function of c-jun N-terminal kinase (JNK) and NFκB in a model of oral tumor cells

The aim of this study is to identify potential gene and protein targets when nuclear factor kappa B (NFkappaB) and c-jun N-terminal kinase (JNK) were inversely expressed in oral tumors. To determine which genes were regulated synergistically by the inverse expression of NFkappaB and JNK, a pathway specific microarray analysis was performed. While either inhibition of NFkappaB or activation of JNK alone was unable to affect the IGFBP6 gene expression in microarray analysis, concomitant increase in JNK activation in the presence of NFkappaB inhibition increased the expression of this gene significantly. Synergistic increase in IGFBP6 gene expression was also confirmed by RT-PCR and Northern blot analysis of transfected cells. Accordingly, the levels of IGFBP6 protein secretion rose synergistically when JNK was over-expressed in NFkappaB knock down cells. In addition, increased expression of JNK in the absence of NFkappaB resulted in a significant induction of cell death in oral tumors when either left untreated or treated with TNF-alpha and TPA. Moreover, when JNK was inhibited by dominant negative JNK (APF), a significant decrease in cell death could be observed in TNF-alpha and TPA treated NFkappaB knock down oral tumors. Therefore, increased induction of IGFBP6 gene or protein expression in oral tumors could be regarded as a potential predictive marker of tumor sensitivity and could be used for prognostic purposes, since a significant correlation could be observed between increased induction of apoptotic cell death and elevated levels of IGFBP6 in these tumors.

Upregulation of DR5 by proteasome inhibitors potently sensitizes glioma cells to TRAIL‐induced apoptosis

This study was undertaken to explore the potential of new therapeutic approaches designed to reactivate cell death pathways in apoptosis-refractory gliomas and to characterize the underlying molecular mechanisms of this reactivation. Here we investigated the sensitivity of a panel of glioma cell lines (U87, U251, U343, U373, MZ-54, and MZ-18) to apoptosis induced by the death receptor ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), TRAIL in combination with gamma irradiation, and TRAIL in combination with proteasome inhibitors (MG132 and epoxomicin). Analysis of these six glioma cell lines revealed drastic differences in their sensitivity to these treatments, with two of the six cell lines revealing no significant induction of cell death in response to TRAIL alone. Interestingly, the proteasome inhibitors MG132 and epoxomicin were capable of potentiating TRAIL-induced apoptosis in TRAIL-sensitive U87 and U251 cells and of reactivating apoptosis in TRAIL-resistant U343 and U373 cells. In contrast, gamma irradiation had no synergistic effects with TRAIL in the two TRAIL-resistant cell lines. RNA interference against death receptor 5 (DR5) revealed that reactivation of TRAIL-induced apoptosis by proteasome inhibitors depended on enhanced transcription and surface expression of DR5. Transient knockdown of the transcription factor GADD153/C/EBP homologous protein and application of the synthetic c-Jun N-terminal kinase inhibitor SP600125 indicated that enhanced DR5 expression occurred independently of GADD153/C/EBP homologous protein, but required activation of the c-Jun N-terminal kinase/c-Jun signaling pathway. Novel therapeutic approaches using TRAIL or agonistic TRAIL receptor antibodies in combination with proteasome inhibitors may represent a promising approach to reactivate apoptosis in therapy-resistant high-grade gliomas.