Significant Increase In Cell Death Research Articles

Abstract 2097: A novel J-series prostamide induces ER stress-mediated apoptosis and upregulates ER oxidoreductase 1 alpha (ERO1α) in human colon cancer cells

Abstract Colon cancer is the third most common cancer and the third leading cause of cancer-related death in the United States. The endoplasmic reticulum (ER) is a cellular organelle responsible for protein synthesis and oxidative folding. ER stress occurs when the protein folding load exceeds the protein folding capacity. Low levels of ER stress promote survival while excessive ER stress causes cell death. An important regulator of the cytotoxic ER stress pathway is the transcription factor, C/EBP homologous protein 10 (CHOP10). It has been shown that CHOP10 regulates the transcription of ER oxidoreductase 1α (ERO1α). ERO1α promotes ER luminal oxidation and under conditions of excessive ER stress releases H2O2 into the cytoplasm, resulting in oxidative stress-mediated apoptosis. Previous studies from our laboratory showed that 15deoxy, Δ12,14 prostamide J2 (15d PMJ2) induced ER stress-mediated apoptosis, leading to a reduction in cell viability in tumorigenic keratinocytes and melanocytes. In addition, 15d PMJ2-induced ER stress-apoptosis was decreased in the presence of ER stress inhibitors, 4-phenylbuterate (PBA) and salubrinal. However, the specific pathway involved in 15d PMJ2-induced ER stress-apoptosis have not been identified. In this study, we hypothesize that 15d PMJ2 causes ER stress-mediated apoptosis in colon tumor cells by activating CHOP10 and its downstream transcriptional target, ERO1α. To examine the anti-proliferative effect of 15d PMJ2, tumorigenic colon cells (HCT116) and non-tumorigenic colon cells (FHC) were treated with different concentrations of 15d PMJ2 or 10µM thapsigargin (TG) for 24 hours and cytotoxicity was measured by lactate dehydrogenase (LDH) assay. A significant increase in cell death was observed in HCT116 cells treated with 5µM 15d PMJ2 and this cytotoxic effect was 3-fold greater in HCT116 compared to FHC cells. Apoptotic measurements showed a significant increase in caspase 3/7 activity and annexin-V positivity following 15d PMJ2 treatment. The expression of CHOP10 was significantly enhanced in the 15d PMJ2-treated group. In addition, 15d PMJ2-induced apoptosis was decreased in the presence of ER stress inhibitors, PBA and salubrinal, suggesting that ER stress is essential for 15d PMJ2-induced apoptosis. Western blot analysis revealed an increase in ERO1α protein expression following 15d PMJ2 treatment. Apoptosis measurements showed a significant inhibition in 15d PMJ2-mediated apoptosis in the presence of ERO1α inhibitor, EN460, suggesting that ERO1α is important for 15d PMJ2-induced apoptosis. These findings suggest that 15d PMJ2-induced apoptosis is mediated via the ER stress pathway. CHOP10 and its transcriptional target, ERO1α, play a potential role in 15d PMJ2-induced ER stress-apoptosis, suggesting that 15d PMJ2 could be a potential anti-neoplastic agent with a unique mechanism for colon cancer. Citation Format: Hussam M. Albassam, Daniel A. Ladin, Rukiyah Van Dross. A novel J-series prostamide induces ER stress-mediated apoptosis and upregulates ER oxidoreductase 1 alpha (ERO1α) in human colon cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2097. doi:10.1158/1538-7445.AM2017-2097

Abstract 4066: The dihydroartemisinin oxime dimer (NSC735847) displays a selective toxicity in colon cancer cells which is potentially mediated by endoplasmic reticulum stress

Abstract Colorectal cancer (CRC) is the third leading cause of cancer-related deaths in the United States. Common chemotherapeutic regimens for CRC include a combination of chemotherapeutic agents to produce synergistic drug activity and reduce adverse effects. However, adverse effects due to lack of selective toxicity is still a major problem with many chemotherapeutic agents. Novel drugs that display tumor selective toxicity are desperately needed. Several studies have shown that artemisinin monomers (Clinically used anti-malarial agent) possess antineoplastic activity with minimal toxicity. Interestingly, artemisinin dimers showed more potent antineoplastic activity compared to the monomers. However, few studies have fully characterized the activity of these molecules. In this study, we tested the antitumor activity of five chemically-synthesized dihydroartemisinin (DHA) dimers using the human colon cancer cell lines, HT29 and HCT116 and the non-tumorigenic colon cell line, FHC. Compared to other tested dimers, the DHA oxime dimer, NSC735847 showed pronounced selective toxicity in HT29 and HCT116 cells. Using MTS cell viability assays, NSC735847caused a preferential reduction in the viability of HT29 and HCT116 colon cancer cells compared to the non-tumorigenic FHC cells. In addition, NSC735847 significantly increased caspase 3/7 activity in HT29 and HCT116 cells but not in FHC cells which suggests that this compound causes selective apoptosis in these colon cancer cell lines. The combination of NSC735847 and the topoisomerase I inhibitor, irinotecan (commonly used chemotherapeutic agent for colorectal cancer) showed synergistic antitumor activity in HT29 cells. The combination of the two drugs caused a significant increase in cell death and caspase 3/7 activity which were greater than those caused by the individual drugs. Previous studies using other tumorigenic cell lines, suggested that NSC735847 induces oxidative and endoplasmic reticulum (ER) stress. To gain insight into the potential mechanisms of anti-colorectal cancer activity of NSC735847, we tested if this compound causes ER stress and/ or oxidative stress in HT29 cells and whether ER stress was required for NSC735847-induced apoptotic cell death. NSC735847 increased the expression of ER stress marker C/EBP homologous protein 10 (CHOP10) in HT29 cells and the use of ER stress inhibitor, salubrinal, significantly decreased NSC735847-induced cell death and apoptosis. Additionally, NSC735847 caused oxidative stress in HT29 cells which was inhibited by pretreatment of the cells with the antioxidant, trolox. The crosstalk between oxidative stress and ER stress in NSC735847-induced apoptosis in colon cancer cells is still under investigation. Our results suggest that NSC735847 causes ER stress in HT29 cells which plays a major role in drug-induced cell death and apoptosis in these cells. Citation Format: Ahmed E. Elhassanny, Eman Soliman, Paul McGuire, Mahmoud ElSohly, Waseem Gul, Rukiyah Van Dross. The dihydroartemisinin oxime dimer (NSC735847) displays a selective toxicity in colon cancer cells which is potentially mediated by endoplasmic reticulum stress [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4066. doi:10.1158/1538-7445.AM2017-4066

Abstract 3178: The p53 tumor suppressor protein paradoxically drives chemo-resistance in human medulloblastoma cells through suppressing the mTOR AKT pathway

Abstract Medulloblastoma (MB) accounts for approximately 25% of childhood brain tumors with 70% of the cases occurring in children under 10. Prognosis for children less than three years old is considerably worse and, due to its destructive effects on the developing nervous system, irradiation is largely avoided in this age group. The p53 pathway is considered a key determinant of anti-tumor responses in many tumors; however, its role in the regulation of cell survival, chemo-sensitivity and chemo-resistance in MB is much less well defined. It has been shown that p53 pathway defects, mutations and nuclear levels increase significantly from MB diagnosis to relapse and correlate with an adverse prognosis. We recently reported on the highly novel and unexpected finding that both the genetic (sh-RNA) and chemical silencing of p53 led to a significant increase in cell death by the drug VMY in MB cell lines with elevated basal p53 (e.g. in D556 cells which express wild type p53 and in DAOY cells that contain mutant p53) as measured by colony forming assays, DNA degradation assays and annexin-V staining, suggesting a surprising commonality in the p53 signaling in both cell lines despite the differences in their p53 status. Equally surprising was the observation that the silencing of p53 in D556 cells enhanced cell death by the clinically used drugs doxorubicin and vincristine. Conversely, we now find that suppressing p53 with sh-RNA in D283 cells, which express much lower levels of wild type p53 compared to D556, resulted in the more classical chemoresistance profile, suggesting a form of p53 ‘addiction’ in D556 and DAOY cells. Mechanistically, we observed that suppressing p53 with sh-RNA in D556 cells treated with VMY or doxorubicin significantly increased the levels of phosphorylated Chk1, S6K, gH2AX, and MDM2. Importantly, phospho-mTOR levels were also significantly increased in the D556/p53 sh-RNA cells and the suppression of mTOR enhanced chemoresistance. These somewhat paradoxical findings suggest that activated Akt/mTOR may induce MB cell death and that suppression of mTOR by p53 enhances chemoresistance. Our data provide new mechanistic insights into the role of p53 in primitive neuroectodermal tumors and may provide new approaches for enhancing the clinical outcome of patients with MB. Citation Format: Aisha Naeem, Muhammad U. Choudhry, Maria L. Avantaggiati, Olga C. Rodriguez, Chris Albanese. The p53 tumor suppressor protein paradoxically drives chemo-resistance in human medulloblastoma cells through suppressing the mTOR AKT pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3178. doi:10.1158/1538-7445.AM2017-3178

Vulnerability to a Metabolic Challenge Following Perinatal Asphyxia Evaluated by Organotypic Cultures: Neonatal Nicotinamide Treatment.

The hypothesis of enhanced vulnerability following perinatal asphyxia was investigated with a protocol combining in vivo and in vitro experiments. Asphyxia-exposed (AS) (by 21min water immersion of foetuses containing uterine horns) and caesarean-delivered control (CS) rat neonates were used at P2-3 for preparing triple organotypic cultures (substantia nigra, neostriatum and neocortex). At DIV 18, cultures were exposed to different concentrations of H2O2 (0.25-45mM), added to the culture medium for 18h. After a 48-h recovery period, the cultures were either assessed for cell viability or for neurochemical phenotype by confocal microscopy. Energy metabolism (ADP/ATP ratio), oxidative stress (GSH/GSSG) and a modified ferric reducing/antioxidant power assay were applied to homogenates of parallel culture series. In CS cultures, the number of dying cells was similar in substantia nigra, neostriatum and neocortex, but it was several times increased in AS cultures evaluated under the same conditions. A H2O2 challenge led to a concentration-dependent increase in cell death (>fourfold after 0.25mM of H2O2) in CS cultures. In AS cultures, a significant increase in cell death was only observed after 0.5mM of H2O2. At higher than 1mM of H2O2 (up to 45mM), cell death increased several times in all cultures, but the effect was still more prominent in CS than in AS cultures. The cell phenotype of dying/alive cells was investigated in formalin-fixed cultures exposed to 0 or 1mM of H2O2, co-labelling for TUNEL (apoptosis), MAP-2 (neuronal phenotype), GFAP (astroglial phenotype) and TH (tyrosine hydroxylase; for dopamine phenotype), counterstaining for DAPI (nuclear staining), also evaluating the effect of a single dose of nicotinamide (0.8nmol/kg, i.p. injected in 100μL, 60min after delivery). Perinatal asphyxia produced a significant increase in the number of DAPI/TUNEL cells/mm3, in substantia nigra and neostriatum. One millimolar of H202 increased the number of DAPI/TUNEL cells/mm3 by ≈twofold in all regions of CS and AS cultures, an effect that was prevented by neonatal nicotinamide treatment. In substantia nigra, the number of MAP-2/TH-positive cells/mm3 was decreased in AS compared to CS cultures, also by 1mM of H202, both in CS and AS cultures, prevented by nicotinamide. In agreement, the number of MAP-2/TUNEL-positive cells/mm3 was increased by 1mM H2O2, both in CS (twofold) and AS (threefold) cultures, prevented by nicotinamide. The number of MAP-2/TH/TUNEL-positive cells/mm3 was only increased in CS (>threefold), but not in AS (1.3-fold) cultures. No TH labelling was observed in neostriatum, but 1mM of H2O2 produced a strong increase in the number of MAP-2/TUNEL-positive cells/mm3, both in CS (>2.9-fold) and AS (>fourfold), decreased by nicotinamide. In neocortex, H2O2 increased the number of MAP-2/TUNEL-positive cells/mm3, both in CS and AS cultures (≈threefold), decreased by nicotinamide. The ADP/ATP ratio was increased in AS culture homogenates (>sixfold), compared to CS homogenates, increased by 1mM of H202, both in CS and AS homogenates. The GSH/GSSG ratio was significantly decreased in AS, compared to CS cultures. One millimolar of H2O2 decreased that ratio in CS and AS homogenates. The present results demonstrate that perinatal asphyxia induces long-term changes in metabolic pathways related to energy and oxidative stress, priming cell vulnerability with both neuronal and glial phenotype. The observed effects were region dependent, being the substantia nigra particularly prone to cell death. Nicotinamide administration in vivo prevented the deleterious effects observed after perinatal asphyxia in vitro, a suitable pharmacological strategy against the deleterious consequences of perinatal asphyxia.

Age-related effect of cell death on fiber morphology and number in tongue muscle.

Multiple pathways may exist for age-related tongue muscle degeneration. Cell death is one mechanism contributing to muscle atrophy and decreased function. We hypothesized with aging, apoptosis, and apoptotic regulators would be increased, and muscle fiber size and number would be reduced in extrinsic tongue muscles. Cell death indices, expression of caspase-3 and Bcl-2, and measures of muscle morphology and number were determined in extrinsic tongue muscles of young and old rats. Significant increases in cell death, caspase-3, and Bcl-2 were observed in all extrinsic tongue muscles along with reductions in muscle fiber number in old rats. We demonstrated that apoptosis indices increase with age in lingual muscles and that alterations in apoptotic regulators may be associated with age-related degeneration in muscle fiber size and number. These observed apoptotic processes may be detrimental to muscle function, and may contribute to degradation of cranial functions with age. Muscle Nerve 57: E29-E37, 2018.

Gold Nanoparticles for BCR-ABL1 Gene Silencing: Improving Tyrosine Kinase Inhibitor Efficacy in Chronic Myeloid Leukemia.

Introduction of tyrosine kinase inhibitors for chronic myeloid leukemia treatment is associated with a 63% probability of maintaining a complete cytogenetic response, meaning that over 30% patients require an alternative methodology to overcome resistance, tolerance, or side effects. Considering the potential of nanotechnology in cancer treatment and the benefits of a combined therapy with imatinib, a nanoconjugate was designed to achieve BCR-ABL1 gene silencing. Gold nanoparticles were functionalized with a single-stranded DNA oligonucleotide that selectively targets the e14a2 BCR-ABL1 transcript expressed by K562 cells. This gold (Au)-nanoconjugate showed great efficacy in gene silencing that induced a significant increase in cell death. Variation of BCL-2 and BAX protein expression, an increase of caspase-3 activity, and apoptotic bodies in cells treated with the nanoconjugate demonstrate its aptitude for inducing apoptosis on K562 BCR-ABL1-expressing cells. Moreover, the combination of the silencing Au-nanoconjugate with imatinib prompted a decrease of imatinib IC50. This Au-nanoconjugate was also capable of inducing the loss of viability of imatinib-resistant K562 cells. This strategy shows that combination of Au-nanoconjugate and imatinib make K562 cells more vulnerable to chemotherapy and that the Au-nanoconjugate alone may overcome imatinib-resistance mechanisms, thus providing an effective treatment for chronic myeloid leukemia patients who exhibit drug tolerance.

Forced expression of Wnt antagonists sFRP1 and WIF1 sensitizes chronic myeloid leukemia cells to tyrosine kinase inhibitors.

Chronic myeloid leukemia is a clonal myeloproliferative disorder that arises from the neoplastic transformation of the hematopoietic stem cell, in which the Wnt/β-catenin signaling pathway has been demonstrated to play an important role in disease progression. However, the role of Wnt signaling antagonists in therapy resistance and disease progression has not been fully investigated. We aimed to study the effects of Wnt/β-catenin pathway antagonists-secreted frizzled-related protein 1 and Wnt inhibitory factor 1-on resistance toward tyrosine kinase inhibitors in chronic myeloid leukemia. Response to tyrosine kinase inhibitors was analyzed in secreted frizzled-related protein 1 and Wnt inhibitory factor 1 stably transfected K562 cells. Experiments were repeated using a tetracycline-inducible expression system, confirming previous results. In addition, response to tyrosine kinase inhibitor treatment was also analyzed using the secreted frizzled-related protein 1 expressing, BCR-ABL positive MEG01 cell line, in the presence and absence of a secreted frizzled-related protein 1 inhibitor. Our data suggests that total cellular β-catenin levels decrease in the presence of secreted frizzled-related protein 1 and Wnt inhibitory factor 1, and a significant increase in cell death after tyrosine kinase inhibitor treatment is observed. On the contrary, when secreted frizzled-related protein 1 is suppressed, total β-catenin levels increase in the cell and the cells become resistant to tyrosine kinase inhibitors. We suggest that Wnt antagonists carry the potential to be exploited in designing new agents and strategies for the advanced and resistant forms of chronic myeloid leukemia.

Efficacy of radiosensitizing doped titania nanoparticles under hypoxia and preparation of an embolic microparticle.

The aim of this study was to develop a manufacturing protocol for large-scale production of doped titania radiosensitizing nanoparticles (NPs) to establish their activity under hypoxia and to produce a multimodal radiosensitizing embolic particle for cancer treatment. We have previously shown that radiosensitizing NPs can be synthesized from titania doped with rare earth elements, especially gadolinium. To translate this technology to the clinic, a crucial step is to find a suitable, scalable, high-throughput method. Herein, we have described the use of flame spray pyrolysis (FSP) to generate NPs from titanium and gadolinium precursors to produce titania NPs doped with 5 at% gadolinium. The NPs were fully characterized, and their capacity to act as radiosensitizers was confirmed by clonogenic assays. The integrity of the NPs in vitro was also ascertained due to the potentially adverse effects of free gadolinium in the body. The activity of the NPs was then studied under hypoxia since this is often a barrier to effective radiotherapy. In vitro radiosensitization experiments were performed with both the hypoxia mimetics deferoxamine and cobalt chloride and also under true hypoxia (oxygen concentration of 0.2%). It was shown that the radiosensitizing NPs were able to cause a significant increase in cell death even after irradiation under hypoxic conditions such as those found in tumors. Subsequently, the synthesized NPs were used to modify polystyrene embolization microparticles. The NPs were sintered to the surface of the microparticles by heating at 230°C for 15 minutes. This resulted in a good coverage of the surface and to generate embolization particles that were shown to be radiosensitizing. Such multimodal particles could therefore result in occlusion of the tumor blood vessels in conjunction with localized reactive oxygen species generation, even under hypoxic conditions such as those found in the center of tumors.

PI3K Inhibitors Synergize with FGFR Inhibitors to Enhance Antitumor Responses in FGFR2mutant Endometrial Cancers.

Improved therapeutic approaches are needed for the treatment of recurrent and metastatic endometrial cancer. Endometrial cancers display hyperactivation of the MAPK and PI3K pathways, the result of somatic aberrations in genes such as FGFR2, KRAS, PTEN, PIK3CA, and PIK3R1 The FGFR2 and PI3K pathways, have emerged as potential therapeutic targets in endometrial cancer. Activation of the PI3K pathway is seen in more than 90% of FGFR2mutant endometrial cancers. This study aimed to examine the efficacy of the pan-FGFR inhibitor BGJ398 with pan-PI3K inhibitors (GDC-0941, BKM120) and the p110α-selective inhibitor BYL719. We assessed synergy in three FGFR2mutant endometrial cancer cell lines (AN3CA, JHUEM2, and MFE296), and the combination of BGJ398 and GDC-0941 or BYL719 showed strong synergy. A significant increase in cell death and decrease in long-term survival was seen when PI3K inhibitors were combined with BGJ398. Importantly, these effects were seen at low concentrations correlating to only partial inhibition of AKT. The combination of BGJ398 and GDC-0941 showed tumor regressions in vivo, whereas each drug alone only showed moderate tumor growth inhibition. BYL719 alone resulted in increased tumor growth of AN3CA xenografts but in combination with BGJ398 resulted in tumor regression in both AN3CA- and JHUEM2-derived xenografts. These data provide evidence that subtherapeutic doses of PI3K inhibitors enhance the efficacy of anti-FGFR therapies, and a combination therapy may represent a superior therapeutic treatment in patients with FGFR2mutant endometrial cancer. Mol Cancer Ther; 16(4); 637-48. ©2017 AACR.

An Investigation of Sex Difference in Microglia Morphology and Function

Microglia are the resident immune cells of the brain. During development microglia progenitor cells migrate from the periphery into the central nervous system. During this critical developmental period, immature microglia differ greatly in their shape and function from those in the adult brain. Immune activation of microglia in the young, developing brain, via neonatal infection, can often lead to long‐term changes in microglial function with associated neuronal and cognitive dysfunction. Furthermore, microglial activation is associated with multiple neurodevelopmental disorders with known or suspected immune etiologies. Such neurological disorders include autism, ADHD, schizophrenia, and cerebral palsy – all exhibiting a strong sex bias in males (Schwarz et al. J. Neurochem., 2012; Bilbo et al. J. Neuroimmune Pharmacol., 2011). Given these rodent and well‐known human epidemiological data, we hypothesize that a hightened level of cytokine expression in developing male rat pups increases their vulnerability to an immune challenge. To test this hypothesis, we removed any variability in the number of microglia cells between males and females and isolated microglia cells from the remainder of the neural cell population. A primary culture of microglial cells was prepared from dissociated hippocampal tissue from P4 male and female rat brains. Microglial cells were selected for by immunoprecipitation (CD11b surface marker), using magnetic beads. Cells were counted and 30,000 cells were plated per well. An immune response was trigged by treating the cells with a 1,000ng/mL Lipopolysaccharide (LPS) dose or Saline (for control) prior to flash freezing and RNA extraction. PCR was used to determine the relative gene expression of pro‐inflammatory cytokines Interleukin‐1 Beta (IL‐1B) and Interleukin‐6 (IL‐6), Toll‐Like Receptor 4 (TLR4), Apoptosis Marker (Cideb1), and Hormone Receptors for estrogen (Esr‐1) and androgen (Ar). IL‐1B and IL‐6 expression for microglial populations had a significant main effect for treatment (F1,78 = 68.41; p < 0.001, η2 = 0.533) and (F1,80 = 91.28; p < 0.001, η2 = 0.467) respectively. TLR4 expression in microglial cells had a significant main effect for treatment (F1,81 = 11.36; p = 0.001, η2 = 0.123) and the other neural cells had a trending main effect for sex (F1,27 = 3.49; p = 0.073, η2 = 0.115). Cideb1 expression in the non‐microglial neural cell population had a significant sex by treatment interaction (F1,27 = 5.49; p = 0.027, η2 = 0.169). And there was no gene expression for either hormone receptor detected on microglial cells. We concluded that there is no sex by treatment differences at a 1000ng/mL LPS dose. However, the possible down‐regulation in TLR4 receptors could indicate that 1000ng/mL is too high of a dose for the isolated cells to tolerate and the extreme immune response washed out smaller differences between sexes. The significant increase in cell death of female non‐microglial cells despite the non‐significant difference in pro‐inflammatory cytokine expression between the sexes could indicate a possible sex‐based difference in the way neurons respond to immune challenges.Support or Funding InformationNIH grant R21 MH101883‐01 to Jaclyn M. Schwarz

Surgery increases cell death and induces changes in gene expression compared with anesthesia alone in the developing piglet brain.

In a range of animal species, exposure of the brain to general anaesthesia without surgery during early infancy may adversely affect its neural and cognitive development. The mechanisms mediating this are complex but include an increase in brain cell death. In humans, attempts to link adverse cognitive development to infantile anaesthesia exposure have yielded ambiguous results. One caveat that may influence the interpretation of human studies is that infants are not exposed to general anaesthesia without surgery, raising the possibility that surgery itself, may contribute to adverse cognitive development. Using piglets, we investigated whether a minor surgical procedure increases cell death and disrupts neuro-developmental and cognitively salient gene transcription in the neonatal brain. We randomly assigned neonatal male piglets to a group who received 6h of 2% isoflurane anaesthesia or a group who received an identical anaesthesia plus 15 mins of surgery designed to replicate an inguinal hernia repair. Compared to anesthesia alone, surgery-induced significant increases in cell death in eight areas of the brain. Using RNAseq data derived from all 12 piglets per group we also identified significant changes in the expression of 181 gene transcripts induced by surgery in the cingulate cortex, pathway analysis of these changes suggests that surgery influences the thrombin, aldosterone, axonal guidance, B cell, ERK-5, eNOS and GABAA signalling pathways. This suggests a number of novel mechanisms by which surgery may influence neural and cognitive development independently or synergistically with the effects of anaesthesia.

Combination of bortezomib and daunorubicin in the induction of apoptosis in T-cell acute lymphoblastic leukemia.

Despite advances in the treatment of T-cell acute lymphoblastic leukemia (T-ALL), the outcome of T-ALL treatment remains unsatisfactory, therefore, more effective treatment is urgently required. The present study examined the cytotoxicities of bortezomib in combination with daunorubicin against human Jurkat and Molt-4 T-ALL cells and primary T-ALL cells. Compared with treatment alone, co-exposure of cells to bortezomib and daunorubicin resulted in a significant increase in cell death in the Jurkat cells, as evidenced by the increased percentage of Annexin V-positive cells, the formation of apoptotic bodies. In addition, the administration sequence of bortezomib and daunorubicin had an effect on cell viability. Treatment with bortezomib followed by daunorubicin treatment was more effective, compared with treatment with daunorubicin followed by bortezomib. Co-treatment with bortezomib and daunorubicin markedly enhanced the activation of caspase-3, -8 and -9, which was reversed by the pan-caspase inhibitor, Z-VAD-FMK. In addition, cotreatment with bortezomib and daunorubicin enhanced the collapse of mitochondrial transmembrane potential and upregulated the proapoptotic protein, B-cell lymphoma 2 (Bcl-2)-interacting mediator of cell death (Bim), but not Bcl-2 or Bcl-extra large. Consistent with this, it was demonstrated that cotreatment of bortezomib and daunorubicin efficiently induced apoptosis in primary T-ALL cells, and cell death was associated with the collapse of mitochondrial transmembrane potential and the upregulation of Bim. Taken together, these findings indicated that the combination of bortezomib and daunorubicin significantly enhanced their apoptosis-inducing effect in T-ALL cells, which may warrant further investigation in preclinical and clinical investigations.

Influence of Arsenic (III), Cadmium (II), Chromium (VI), Mercury (II), and Lead (II) Ions on Human Triple Negative Breast Cancer (HCC1806) Cell Cytotoxicity and Cell Viability

The hazardous consequences of heavy metal ions (HMIs) on human health necessitate the immediate need to probe fundamentally the interactions and cytotoxic effects of HMIs on humans. This study investigated the influence of five toxic HMIs (arsenic (As (III)), cadmium (Cd (II)), chromium (Cr (VI)), mercury (Hg (II)), and lead (Pb (II))) on human TNBC (HCC 1806) cell viability using optical microscopy, trypan blue dye-exclusion assays, and flow cytometry. The TNBC cells were exposed to varying concentrations of HMIs for 24 and 48 hours. We evaluated the influence of the concentrations and duration of HMIs exposure on TNBC cell viability. Light microscopy, cell viability assays, revealed that after 48-hour treatment of TNBC cells with 1 x 10 -5 M of As (III), Cd (II), Hg (II), Cr (IV), and Pb (II) resulted in cell viabilities of 23%, 34%, 35%, 56%, 91% respectively, suggesting that As (III) has the greatest cytotoxicity (77% cell death) while Pb (II) showed the least (9% cell death). Furthermore, flow cytometry revealed that while Pb (II), As (III) and Cr (IV) had significant increases in cell death, Hg (II) caused a G1 arrest. Together, this study revealed that HMIs cause a differential cytotoxic effect on TNBC cells and suggest that they may have very different genotoxic targets and implications in their mutagenic potential.

Reduction of Mitochondrial Membrane Potential Leads to Enhancement of Type-II CD20-Antibody Cytotoxicity in Diffuse Large B-Cell Lymphoma

Diffuse Large B Cell Lymphoma (DLBCL) is the most prevalent non-Hodgkin lymphoma (NHL) in adults. Since the addition of the Type I anti-CD20 antibody Rituximab to chemotherapy, the overall survival of NHL patients has improved dramatically compared to the pre-Rituximab era. DLBCL however, has the worst survival rates out of all NHLs with an average 5-year survival of 55%. Unfortunately 40% of all DLBCL patients relapse within 2 years, and those that relapse or have refractory disease tend not to respond well to antibody-based salvage therapies. Since the discovery and utilisation of Rituximab, many have tried to enhance the efficacy of anti-CD20 antibodies in order to improve first-line treatment of DLBCL, leading to the evolution of Type II humanised anti-CD20 antibodies.The complete biological role of CD20 remains unclear, however it has been shown to act as part of an ion channel complex that is a component of the store operated calcium (Ca2+) system. This complex has the ability to facilitate mitochondrial membrane permeabilisation, resulting in reduced mitochondrial function.In order to investigate the effect of Type I- and Type II- anti-CD20 antibodies on mitochondrial function, we established a panel of 4 DLBCL cell lines. We used the XF Seahorse Mito Stress Test to reveal bioenergetic profiles of the cell lines before and after treatment with a panel of Type I and Type II anti-CD20 antibodies (2 Type-I and 2 Type-II anti-CD20 antibodies for each cell line). Basal oxidative phosphorylation (OxPhos), ATP production, and maximal and spare respiratory capacity of each sample were calculated as a measure of mitochondrial function. Next we used Metformin, a well-established inhibitor of oxidative phosphorylation to reduce the mitochondrial membrane potential (MMP) across our panel of cell lines. We confirmed MMP reduction by staining cells with JC-1, a chameleon dye used as an indicator of MMP and analysed samples using flow cytometry. We then used the XF Seahorse Mito Stress Test, this time to assess how combining each CD20-antibody with an OxPhos inhibitor effects mitochondrial function (10 conditions for each cell line). Finally, we used the same conditions to conduct clonogenic survival assays to see whether cytotoxicity of Type-I or Type-II anti-CD20 antibodies could be enhanced.We have observed that treatment with anti-CD20 antibodies results in a significant increase in the maximal respiratory capacity of our panel of cell lines. Conversely, pharmacological inhibition of oxidative phosphorylation causes a significant reduction in basal oxidative phosphorylation as well as a reduction in the maximal respiratory capacity of the cell lines in our panel. We also show that treatment combining an OxPhos inhibitor with either Type-I or Type-II CD20-antibodies prevents the increase in maximal respiratory capacity observed with CD20-antibody treatment alone. When analysing the clonogenic survival of cell lines we have found that only the cytotoxicity of Type-II anti-CD20 antibodies is enhanced by simultaneously treating cell lines with Metformin. We also used Annexin V/PI staining to assess cell death and show that inhibiting oxidative phosphorylation in conjunction with CD20-antibody treatment does not result in a significant increase in cell death across our panel of cell lines.Our data indicate for the first time that when cells are treated with CD20-antibodies they increase their maximal mitochondrial respiratory capacity to compensate for reduced basal mitochondrial function. We also show that inhibition of oxidative phosphorylation disables the cells from being able to compensate for the reduced mitochondrial function that is caused by CD20-antibody treatment. Importantly our data show that the reduction of mitochondrial function caused by combining Metformin with Type-II CD20 antibodies leads to a significant reduction in clonogenicity. We believe that understanding the mechanism of the inhibition of mitochondrial function will allow us to establish effective treatment combinations to significantly improve the efficacy of anti-CD20 antibody therapy in DLBCL. DisclosuresNo relevant conflicts of interest to declare.

Isoflurane Exposure Induces Cell Death, Microglial Activation and Modifies the Expression of Genes Supporting Neurodevelopment and Cognitive Function in the Male Newborn Piglet Brain.

Exposure of the brain to general anesthesia during early infancy may adversely affect its neural and cognitive development. The mechanisms mediating this are complex, incompletely understood and may be sexually dimorphic, but include developmentally inappropriate apoptosis, inflammation and a disruption to cognitively salient gene expression. We investigated the effects of a 6h isoflurane exposure on cell death, microglial activation and gene expression in the male neonatal piglet brain. Piglets (n = 6) were randomised to: (i) naive controls or (ii) 6h isoflurane. Cell death (TUNEL and caspase-3) and microglial activation were recorded in 7 brain regions. Changes in gene expression (microarray and qPCR) were assessed in the cingulate cortex. Electroencephalography (EEG) was recorded throughout. Isoflurane anesthesia induced significant increases in cell death in the cingulate and insular cortices, caudate nucleus, thalamus, putamen, internal capsule, periventricular white matter and hippocampus. Dying cells included both neurons and oligodendrocytes. Significantly, microglial activation was observed in the insula, pyriform, hippocampus, internal capsule, caudate and thalamus. Isoflurane induced significant disruption to the expression of 79 gene transcripts, of these 26 are important for the control of transcription and 23 are important for the mediation of neural plasticity, memory formation and recall. Our observations confirm that isoflurane increases apoptosis and inflammatory responses in the neonatal piglet brain but also suggests novel additional mechanisms by which isoflurane may induce adverse neural and cognitive development by disrupting the expression of genes mediating activity dependent development of neural circuits, the predictive adaptive responses of the brain, memory formation and recall.

Effects of perfluorinated alkyl acids on cellular responses of MCF-10A mammary epithelial cells in monolayers and on acini formation in vitro

Perfluorinated alkyl acids (PFAAs) are stable chemicals detected in tissue and serum from various species, including humans, and have been linked to adverse health outcomes. Experimental PFAA exposure in rodents has been associated with changes in mammary gland development. The estrogen receptor (ER)-negative human breast epithelial cell line, MCF-10A, can be grown as monolayer, but also has the ability to form three-dimensional acini in vitro, reflecting aspects of mammary glandular morphogenesis. Cells were exposed to five different PFAAs, perfluorooctane sulfonate (PFOS), perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), perfluorodecanoic acid (PFDA), and perfluoroundecanoic acid (PFUnDA), both in monolayer and acini cultures. In monolayer cultures only the higher concentrations of PFOS, PFNA and PFDA (400–500μM) caused a significant increase in cell death, whereas PFOA and PFUnDA had no effect. Normal acini maturation was negatively impacted by PFOS, PFNA and PFDA already at the lowest concentration tested (0.6μM). Observed effects included loss of organization of the cell clusters and absence of a hollow lumen. Overall, this study demonstrated that PFAAs can interfere with cellular events related to normal development of glandular breast tissue through ER-independent mechanisms.

Extracorporeal photopheresis as an immunomodulatory agent: Haematocrit-dependent effects on natural killer cells.

The GvHD is a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT). Extracorporeal photopheresis (ECP) represents an alternative therapeutic strategy to immunosuppressive therapy. Although ECP is used since 1990s, the mechanism of action has not yet been completely clarified. We analyzed cells collected from 20 ECP procedures of 4 patients affected by chronic GvHD and, for comparison, Peripheral Blood Mononuclear Cells (PBMCs) of 10 healthy donors undergoing from same type of photochemiotherapy, evaluating by flow cytometry, the effects before and after photoactivation with 8-MOP. The analysis showed a significant increase in cell death after ECP in particular in CD4 T lymphocytes as described in literature correlated with haematocrit value. Most interesting data emerge from the analysis of cytotoxic activity of NK cells, using flow cytometry analysis of surface expression of CD107a in the presence of target cells (K562). In all analyzed samples it was possible to document a statistically significant reduction of the cytotoxic activity of NK cells after photoactivation. The decrease of the cytotoxic activity was related to hematocrit value of leukoapheresis: in fact, lower HCT values were associated with a more marked reduction of cytotoxic activity. The study confirms literature data about the increase of cellular mortality induce by ECP. Furthermore, for the first time it is demonstrated that the ECP exerts a marked and significant inhibitory effect on the cytotoxic activity of NK cells. Our study suggests that lower values of hematocrit are associated with better treatment outcome.

Blocking Interleukin-4 Receptor α Using Polyethylene Glycol Functionalized Superparamagnetic Iron Oxide Nanocarriers to Inhibit Breast Cancer Cell Proliferation.

PurposeThe specific targeting of interleukin-4 receptor α (IL4Rα) receptor offers a promising therapeutic approach for inhibition of tumor cell progression in breast cancer patients. In the current study, the in vitro efficacy of superparamagnetic iron oxide nanoparticles conjugated with anti-IL4Rα blocking antibodies (SPION-IL4Rα) via polyethylene glycol polymers was evaluated in 4T1 breast cancer cells.Materials and MethodsCell viability, reactive oxygen species generation, and apoptosis frequency were assessed in vitro in 4T1 cancer cell lines following exposure to SPION-IL4Rα alone or combined with doxorubicin. In addition, immunofluorescence assessments and fluorimetrywere performed to confirm the specific targeting and interaction of the developed nanocarriers with IL4Rα receptors in breast cancer cells.ResultsBlocking of IL4Rα receptors caused a significant decrease in cell viability and induced apoptosis in 4T1 cells. In addition, combined treatment with SPION-IL4Rα+doxorubicin caused significant increases in cell death, apoptosis, and oxidative stress compared to either SPION-IL4Rα or doxorubicin alone, indicating the enhanced therapeutic efficacy of this combination. The decrease in fluorescence intensity upon immunofluorescence and fluorimetry assays combined with increased viability and decreased apoptosis following the blocking of IL4Rα receptors confirmed the successful binding of the synthesized nanocarriers to the target sites on murine 4T1 breast cancerous cells.ConclusionThese results suggest that SPION-IL4Rα nanocarriers might be used for successfulreduction of tumor growth and inhibition of progression of metastasis in vivo.

Characterization of Pancreatic Cancer Cell Thermal Response to Heat Ablation or Cryoablation.

One of the most lethal carcinomas is pancreatic cancer. As standard treatment using chemotherapy and radiation has shown limited success, thermal regimens (cryotherapy or heat ablation) are emerging as viable alternatives. Although promising, our understanding of pancreatic cancer response to thermal ablation remains limited. In this study, we investigated the thermal responses of 2 pancreatic cancer cell lines in an effort to identify the minimum lethal temperature needed for complete cell death to provide guidance for in vivo applications. PANC-1 and BxPC-3 were frozen (-10°C to -25°C) or heated (45°C-50°C) in single and repeated exposure regimes. Posttreatment survival and recovery were analyzed using alamarBlue assay over a 7-day interval. Modes of cell death were assessed using fluorescence microscopy (calcein acetoxymethyl ester/propidium iodide) and flow cytometry (YO-PRO-1/propidium iodide). Freezing to -10°C resulted in minimal cell death. Exposure to -15°C had a mild impact on PANC-1 survival (93%), whereas BxPC-3 was more severely damaged (33%). Exposure to -20°C caused a significant reduction in viability (PANC-1 = 23%; BxPC-3 = 2%) whereas -25°C yielded complete death. Double freezing exposure was more effective than single exposure. Repeat exposure to -15°C resulted in complete death of BxPC-3, whereas -20°C severely impacted PANC-1 (7%). Heating to 45°C resulted in minimum cell death. Exposure to 48°C yielded a slight increase in cell loss (PANC-1 = 85%; BxPC-3 = 98%). Exposure to 50°C caused a significant decline (PANC-1 = 70%; BxPC-3 = 9%) with continued deterioration to 0%. Double heating to 45°C resulted in similar effects observed in single exposures, whereas repeated 48°C resulted in significant increases in cell death (PANC-1 = 68%; BxPC-3 = 29%). In conclusion, we observed that pancreatic cancer cells were completely destroyed at temperatures <-25°C or >50°C using single thermal exposures. Repeated exposures resulted in increased cell death at less extreme temperatures. Our data suggest that thermal ablation strategies (heat or cryoablation) may represent a viable technique for the treatment of pancreatic cancer.

Connexin 43 Acts as a Proapoptotic Modulator in Cisplatin-Induced Auditory Cell Death.

Gap junction coupling is known to play a role in intercellular communication by the Good Samaritan effect or bystander effect. Nonjunctional connexins (Cxs) may also play certain gap junction-independent roles in cell death or survival. The purpose of the present study was to investigate the role of junctional and nonjunctional Cxs in ototoxic drug-induced auditory cell death by focusing on Cx43 in the cochlea. Nonjunctional Cx43 conditions were prepared by low confluence culture (5 × 103/cm2) or a trafficking inhibitor, brefeldin A (BFA), in auditory cells, and short lengthened Cx43s with amino-terminal (NT; amino acids 1-256) or carboxy-terminal (CT; amino acids 257-382) were transfected into Cx-deficient HeLa cells to avoid gap junction formation. Knockdown of nonchannel Cx43 (small interfering RNA [siRNA]) inhibited Cis-diamminedichloroplatinum (cisplatin)-induced cell death regardless of gap junction formation; however, a gap junction blocker, 18 alpha-glycyrrhetinic acid (18α-GA), showed inhibitory effect only under the junctional condition. BFA did not show any additive influence on the inhibitory effect of siRNA Cx43. Shortened Cx43-transfected HeLa cells also resulted in a significant increase in cell death under cisplatin. In the animal studies with cisplatin-treated rats, hearing thresholds of auditory brainstem response were significantly preserved by a gap junction blocker, carbenoxolone, showing much more preserved stereocilia of hair cells in scanning electron microscopic findings. Innovation and Conclusion: Cx43 plays a proapoptotic role in cisplatin-induced auditory cell death in both junctional and nonjunctional conditions. Targeting the Cx-mediated signaling control may be helpful in designing new therapeutic strategies for drug-induced ototoxicity. Antioxid. Redox Signal. 25, 623-636.