Terms Of Growth Inhibition Research Articles

Duckweed biomarkers for identifying toxic water contaminants?

Surface or ground waters can be contaminated with numerous toxic substances. The duckweeds Lemna minor and Lemna gibba are widely used for assaying waterborne toxicity to higher plants in terms of growth inhibition and photosynthetic pigment reduction. These tests cannot, however, in themselves determine the nature of the agents responsible for toxicity. Morphological, developmental, physiological, biochemical, and genetic responses of duckweeds to exposure to toxic water contaminants constitute biomarkers of toxic effect. In principle, the very detection of these biomarkers should enable the contaminants having elicited them (and being responsible for the toxicity) to be identified. However, in practice, this is severely compromised by insufficient specificity of biomarkers for their corresponding toxicants and by the lack of documentation of biomarker/toxin relationships. The present contribution illustrates the difficulties of using known water contaminant-related duckweed biomarkers to identify toxins, and discusses possibilities for achieving this goal.

Abstract P2-07-03: Systematic characterization of kinase inhibitors reveals heterogeneity in responses by class and cell line

Abstract Several publications have addressed concerns surrounding drug response screens by pointing out sources of variability and by presenting recommendations for better experimental methods and more robust analytical approaches. In the presented profiling effort, we integrated the latest advances in drug response measurement and focused on data diversity and quality rather than on breadth. We selected 32 breast cancer cell lines with a strong bias towards triple negative lines as well as 4 cell lines established from relevant patient-derived xenografts. We evaluated a panel of clinically relevant kinase inhibitors using a microscopy-based dose response assay to measure drug potency, and to quantify drug efficacy in terms of growth inhibition (GR metrics) and cell death. The use of the GR metrics to quantify drug sensitivity enabled us to identify and study differences between cytostatic and cytotoxic responses. This systematic dose response dataset is complemented by measurements of baseline transcript expression levels by mRNAseq, quantification of absolute abundance of ˜12,000 proteins, and relative phosphoprotein levels by shotgun mass spectrometry across all cell lines. Additionally, the baseline activity of transcription factors and kinases were inferred from the mRNA (using VIPER) and phosphoprotein (using kinase enrichment analysis) data, respectively. The complementarity of these multi-omics data has allowed us to address questions about the landscape of breast cancer cell lines such as: Where do the patient-derived lines lay relative to the conventional cell lines? How consistent are the landscapes defined by each dataset? How does integration across datasets provide mechanistic insight into signaling pathways that are active in each cancer subtypes? The measured and inferred baseline data were used to build predictors of the observed drug responses with the goal of identifying the biological processes responsible for the differences in sensitivity across drugs and cell lines. Overall the dataset that has been collected is a valuable resource for understanding drug response in triple negative breast cancer, and the molecular mechanisms that influence it. Citation Format: Mills CE, Subramanian K, Hafner M, Chung M, Boswell SA, Everley RA, Juric D, Sorger PK. Systematic characterization of kinase inhibitors reveals heterogeneity in responses by class and cell line [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-07-03.

Targeting PLK1 overcomes T-DM1 resistance via CDK1-dependent phosphorylation and inactivation of Bcl-2/xL in HER2-positive breast cancer.

Trastuzumab-refractory, HER2 (human epidermal growth factor receptor 2)-positive breast cancer is commonly treated with trastuzumab emtansine (T-DM1), an antibody-drug conjugate of trastuzumab and the microtubule-targeting agent, DM1. However, drug response reduces greatly over time due to acquisition of resistance whose molecular mechanisms are mostly unknown. Here, we uncovered a novel mechanism of resistance against T-DM1 by combining whole transcriptome sequencing (RNA-Seq), proteomics and a targeted small interfering RNA (siRNA) sensitization screen for molecular level analysis of acquired and de novo T-DM1-resistant models of HER2-overexpressing breast cancer. We identified Polo-like kinase 1 (PLK1), a mitotic kinase, as a resistance mediator whose genomic as well as pharmacological inhibition restored drug sensitivity. Both acquired and de novo resistant models exhibited synergistic growth inhibition upon combination of T-DM1 with a selective PLK1 inhibitor, volasertib, at a wide concentration range of the two drugs. Mechanistically, T-DM1 sensitization upon PLK1 inhibition with volasertib was initiated by a spindle assembly checkpoint (SAC)-dependent mitotic arrest, leading to caspase activation, followed by DNA damage through CDK1-dependent phosphorylation and inactivation of Bcl-2/xL. Furthermore, we showed that Ser70 phosphorylation of Bcl-2 directly regulates apoptosis by disrupting the binding to and sequestration of the pro-apoptotic protein Bim. Importantly, T-DM1 resistance signature or PLK1 expression correlated with cell cycle progression and DNA repair, and predicted a lower sensitivity to taxane/trastuzumab combination in HER2-positive breast cancer patients. Finally, volasertib in combination with T-DM1 greatly synergized in models of T-DM1 resistance in terms of growth inhibition both in three dimensional (3D) cell culture and in vivo. Altogether, our results provide promising pre-clinical evidence for potential testing of T-DM1/volasertib combination in T-DM1 refractory HER2-positive breast cancer patients for whom there is currently no treatment available.

TiO2 nanoparticles in seawater: Aggregation and interactions with the green alga Dunaliella tertiolecta

Titanium dioxide nanoparticles (TiO2 NPs) have been widely employed in industrial applications, thus rising concern about their impact in the aquatic environment. In this study we investigated the chemical behaviour of TiO2 NPs in the culture medium and its effect on the green alga Dunaliella tertiolecta, in terms of growth inhibition, oxidative stress, ROS (Reactive Oxygen Species) accumulation and chlorophyll content. In addition, the influence of exopolymeric substances (EPS) excreted by the microalgae on the stability of NPs has been evaluated. The physicochemical characterization showed a high propensity of TiO2 NPs to form micrometric-sized aggregates within 30min, large enough to partially settle to the bottom of the test vessel. Indeed, an increasing amount of TiO2 particles settled out with time, but the presence of EPS seemed to mitigate this behaviour in the first 6h of exposure where the main effects in D. tertiolecta were observed. TiO2 NPs did not inhibit the 72-h growth rate of D. tertiolecta, nor affected the cellular chlorophyll concentration in the range 0.01–10mgL−1. The time-course of ROS production showed an initial transient increase of ROS in TiO2 NP-exposed algae compared to the control, concomitant with an enhancement of catalase activity. Interestingly, intracellular ROS was a small fraction of total ROS, the highest amount being extracellular. The occurrence of cell-mediated chemical transformations of TiO2 NPs in the external medium, related to the presence of EPS, has been evaluated. Our results showed that carbohydrates were the major component of EPS, whereas proteins of medium molecular weight (20–80kDa) were preferentially bound to TiO2 NPs, likely influencing their biological fate.

Expression of K1 Toxin Derivatives in Saccharomyces cerevisiae Mimics Treatment with Exogenous Toxin and Provides a Useful Tool for Elucidating K1 Mechanisms of Action and Immunity.

Killer toxin K1 is a heterodimeric protein toxin secreted by Saccharomyces cerevisiae strains infected with the M1 double-stranded RNA ‘killer’ virus. After binding to a primary receptor at the level of the cell wall, K1 interacts with its secondary plasma membrane receptor Kre1p, eventually leading to an ionophoric disruption of membrane function. Although it has been under investigation for decades, neither the particular mechanisms leading to toxicity nor those leading to immunity have been elucidated. In this study, we constructed derivatives of the K1α subunit and expressed them in sensitive yeast cells. We show that these derivatives are able to mimic the action of externally applied K1 toxin in terms of growth inhibition and pore formation within the membrane, leading to a suicidal phenotype that could be abolished by co-expression of the toxin precursor, confirming a mechanistic similarity of external and internal toxin action. The derivatives were successfully used to investigate a null mutant completely resistant to externally applied toxin. They provide a valuable tool for the identification of so far unknown gene products involved in K1 toxin action and/or immunity.

Impact of Cadmium and Selenium Exposure on Trace Elements, Fatty Acids and Oxidative stress in Lepidium sativum

The objective of this work was to examine possible effects that the exposure to cadmium and selenium might have in Lepidium sativum. Nine hydroponic cultures were obtained, according to the three level factorial design that covered plants exposure to 0, 0.5, 2.0 mg L-1 of cadmium (as cadmium chloride) and selenium (as sodium selenite). Shoots growth was evaluated during two weeks after germination and the following parameters were measured in the biomass: Cd, Se, Mn, Zn, Cu, Mo, fatty acids and malondialdehyde. The results obtained indicate that selenium is capable to counterbalance the adverse effects of cadmium in terms of growth inhibition, decreased concentration levels of essential micronutrients and oxidative damage;however, such protective role is apparently restricted by the concentration levels of two elements in the growth medium.

Abstract 4711: Immunomodulatory effects of an EphA2-targeted docetaxel antibody-directed nanotherapeutic and synergistic combination with PD-1 inhibitor

Abstract The efficacy of the current class of PD-1/PD-L1 antagonists can be limited by the immunogenicity of the tumor microenvironment. Studies showed that some chemotherapeutic agents including taxanes and anthracyclines can increase immunogenicity, resulting in therapeutic synergy with immune checkpoint inhibitors. In particular, treatment with a taxane has been shown to increase the recruitment of CTLs and decrease immunosuppressive cells such as MDSCs and T-regs. Additionally, the immune-modulatory activity of paclitaxel has been shown to increase with prolonged exposure of the taxane at the tumor level, achieved through metronomic dosing. MM-310 is an Ephrin Receptor A2 (EphA2)-targeted antibody-directed nanotherapeutic (ADN) that encapsulates a docetaxel prodrug. Preclinically, MM-310 leads to prolonged exposure of docetaxel at the tumor level, while lowering systemic exposure to bioavailable docetaxel, and thus decreasing not only dose-limiting neutropenia but also the killing of circulating lymphocytes potentially critical to anti-PD1/PDL1 activity relative to free docetaxel. Taken together, we hypothesize that MM-310 can synergistically combine with anti-PD-1 therapy. In this study, we evaluated the potential combination of MM-310 and a murine anti-PD-1 Ab in the treatment of several syngeneic mouse tumor models. The tumor lines EMT-6, CT-26, and LLC were selected to provide a range of sensitivity to both docetaxel and anti-PD-1. In vivo activity studies and immune-phenotype studies were performed comparing MM-310+anti-PD-1 combination to the monotherapies. MM-310 administration was initiated two days prior to anti-PD-1 therapy and consisted of four weekly doses, while anti-PD-1 was dosed twice weekly for four weeks. The response to MM-310 or anti-PD-1 as monotherapies varied between the models. LLC was unresponsive to anti-PD-1 and poorly responsive to MM-310, CT-26 was poorly responsive to both anti-PD-1 and MM-310, while EMT-6 responded moderately to anti-PD-1 with tumor stasis and well to MM-310, achieving tumor regression. In all models, however, MM-310 given in combination with anti-PD-1 outperformed controls and both monotherapy arms in terms of growth inhibition and tumor regression rate. In the EMT-6 model, combination treatment resulted in durable complete regressions in 6/10 mice when compared to 2/10 and 0/10 for MM-310 and anti-PD-1 monotherapies respectively. In MM-310 and MM-310+anti-PD-1 groups, re-challenge of mice with the same tumor cells, at 10 weeks post treatment interruption, did not lead to tumor growth, suggesting that treatment with anti-PD1+MM-310 promotes the development of a memory response against the tumor antigen. In conclusion, the novel combination of MM-310, an EphA2-targeted docetaxel ADN with an anti-PD-1 antibody, is highly active in syngeneic tumor models, and represents a promising strategy for the treatment of cancer. Citation Format: Andrew J. Sawyer, Anne-Sophie Dugast, Lia Luus, Zhaohua Richard Huang, Suresh K. Tipparaju, Dmitri B. Kirpotin, Daryl C. Drummond, Walid S. Kamoun. Immunomodulatory effects of an EphA2-targeted docetaxel antibody-directed nanotherapeutic and synergistic combination with PD-1 inhibitor [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 4711. doi:10.1158/1538-7445.AM2017-4711

Abstract 2385: Systematic characterization of kinase inhibitors reveals heterogeneity in responses by class and cell line

Abstract Several publications have addressed concerns surrounding drug response screens by pointing out sources of variability and by presenting recommendations for better experimental methods and more robust analytical approaches. In the presented profiling effort, we integrated the latest advances in drug response measurement and focused on data diversity and quality rather than on breadth. We selected 32 breast cancer cell lines with a strong bias towards triple negative lines as well as 4 cell lines established from relevant patient-derived xenografts. We evaluated a panel of clinically relevant kinase inhibitors using a microscopy-based drug response assay to measure drug potency, and to quantify drug efficacy in terms of growth inhibition (GR metrics) and cell death. The use of the GR metrics to quantify drug sensitivity enabled us to identify and study differences between cytostatic and cytotoxic responses. This systematic dose response dataset is complemented by measurements of baseline mRNA expression levels by RNAseq and endogenous protein levels by shotgun mass spectrometry across all cell lines. The completeness and controlled conditions under which these data sets were collected provide confidence in their integration. The baseline RNA and protein expression levels were used to build predictors of the measured drug responses with the goal of identifying the biological processes responsible for the differences in sensitivity across drugs and cell lines. Differences in the phenotypic responses of cell lines to kinase inhibitors with the same nominal targets have been investigated, and associated with variable inhibitor polypharmacology. Citation Format: Caitlin E. Mills, Marc Hafner, Dejan Juric, Peter K. Sorger. Systematic characterization of kinase inhibitors reveals heterogeneity in responses by class and cell line [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 2385. doi:10.1158/1538-7445.AM2017-2385

Abstract P4-08-02: Integration of transcriptomic, proteomic and drug response data in triple negative breast cancer cell lines and PDX models

Abstract Drug response screens on panels of cell lines aimed at identifying markers of sensitivity or resistance have been limited in their successes. Unfortunately, the recent release of many such studies has been accompanied by concerns surrounding reproducibility. Since then, several publications have addressed these concerns by pointing out sources of variability and by suggesting better experimental methods as well as more robust analytical approaches. In the presented profiling effort, we integrated the latest advances in drug response measurement and focused on data diversity and quality rather than on breadth. We selected 32 breast cancer cell lines with a strong bias towards triple negative lines as well as 4 cell lines established from relevant patient-derived xenografts. We used high content microscopy to assay the phenotypic responses of the cell lines to a panel of 34 drugs made up largely of kinase inhibitors currently in the clinic along with some standard of care chemotherapeutics. The microscopy based drug response assay allowed us to measure drug potency, and to quantify the efficacy of the drugs in terms of growth inhibition and cell death. For the same cell lines, we used RNAseq to measure basal mRNA expression levels and shotgun mass spectrometry to measure endogenous protein levels. The completeness and controlled conditions under which these data sets were collected provide confidence in their integration. The complementarity of these multi-omics data has allowed us to address questions about the landscape of, particularly triple negative, breast cancer cell lines. Such questions include: where do the patient-derived lines lay among the established cell lines? and how different are the landscapes defined by drug response phenotypes, mRNA expression, and protein levels? We used network-based algorithms to identify eigenstates of signaling pathways related to genomic events, and further explored these states in the TCGA data. At the level of drug response, we have focused on important questions related to the clinical use of kinase inhibitors. In particular, we compared various CDK inhibitors in an effort to identify markers that are informative of response potency and efficacy. We have also looked at variability of the responses of the cell lines studied to multiple PI3K inhibitors that either target specific isoforms or all isoforms. Overall the data set that has been collected is a valuable resource for understanding drug response in triple negative breast cancer, and the transcriptomic and proteomic factors that influence it. Citation Format: Mills CE, Hafner MA, Sorger PK. Integration of transcriptomic, proteomic and drug response data in triple negative breast cancer cell lines and PDX models [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-08-02.

Aspirin plus sorafenib potentiates cisplatin cytotoxicity in resistant head and neck cancer cells through xCT inhibition

The nonsteroidal anti-inflammatory drug aspirin and the multikinase inhibitor sorafenib have both shown experimental and clinical anticancer activities. The present study investigated whether aspirin and sorafenib synergize to potentiate cisplatin treatment in resistant head and neck cancer (HNC) cells. The effects of aspirin, sorafenib and cisplatin, and combinations thereof were assessed by measuring cell viability, death, glutathione (GSH) and reactive oxygen species (ROS) levels, protein and mRNA expression, genetic inhibition and overexpression of cystine–glutamate antiporter (xCT) and tumor xenograft mouse models. Even at low concentrations, aspirin plus sorafenib synergized to induce cell death of cisplatin-resistant HNC cells. The combination of aspirin and sorafenib induced xCT inhibition, GSH depletion, and ROS accumulation in cancer cells. Genetic and pharmacological inhibition of xCT potentiated the cytotoxic effects of aspirin plus sorafenib; this effect was diminished by xCT overexpression. Low-dose aspirin plus sorafenib enhanced the cytotoxicity of cisplatin in resistant HNC cells through xCT inhibition and oxidant and DNA damage. The in vivo effects of aspirin plus sorafenib on cisplatin therapy were also confirmed in resistant HNC xenograft models, in terms of growth inhibition, GSH depletion, and increased γH2AX formation and apoptosis in tumors. Aspirin and sorafenib synergize to potentiate the cytotoxicity of cisplatin in resistant HNC cells. This therapeutic strategy may help to eliminate resistant HNC.

Synthetic pyrethroids (Type II) and freshwater fish culture: Perils and mitigations

As a new class of agricultural insecticides, synthetic pyrethroids are widely used to control insect pests. Synthetic pyrethroids have been shown to enter the aquatic environment from agricultural runoff or drift from aerial and ground-based spraying applications posing threat to fishes which are less tolerant to pesticides through direct exposure. These insecticides interfere with the sodium channel of the nervous system resulting in prolonged sodium tail current. Widespread application of these chemicals has warranted the attention of the ecologist to understand the impact of these chemicals on the aquatic environment. In this perspective, an updated account of toxicological evaluation of three type II synthetic pyrethroids, viz. deltamethrin, cypermethrin and fenvalerate in terms of their physico-chemical, metabolic, hematological, histological, behavioral and reproductive aspects with respect to the fishes has been presented which may be useful for policy makers, academics, environmental scientists and agricultural professionals needing ready access to this information. The aim of the present synoptic literature appraisal was to summarize the main effect of current use, type II synthetic pyrethroids (deltamethrin, cypermethrin and fenvalerate) on aquatic environment due to their persistence and accumulation. This article will focus on non-target organisms in inland fresh water environment with special reference to fin fishes and will critically evaluate the toxicity of these pyrethroids in terms of growth inhibition, metabolic disorders, neurotoxicity, reproductive failure, enzymatic dysfunction, haematological alterations, and tissue damages. The rationalized information in this milieu may be useful in ecological risk evaluation and human health management as fish serves as an important bio-indicator for aquatic systems health.

Salinity-dependent toxicities of zinc oxide nanoparticles to the marine diatom Thalassiosira pseudonana

This study comprehensively investigated the influences of salinity, exposure concentration and time on the aggregate size, surface charge and dissolution of zinc oxide nanoparticles (ZnO–NPs; 20nm) in seawater, and examined the interacting effect of salinity and waterborne exposure of ZnO–NPs on the marine diatom Thalassiosira pseudonana for 96h. We found that aggregate sizes of ZnO–NPs significantly increased with increasing salinity, but generally decreased with increasing exposure concentration. Ion release decreased with increasing salinity, whereas the surface charge of the particles was not affected by salinity. The increased aggregate size and decreased ion release with increasing salinity, and consequently lower concentration of bioavailable zinc ions, resulted in decreased toxicity of ZnO–NPs at higher salinity in general in terms of growth inhibition (IC50) and chlorophyll fluorescence (EC50 – ФPo and EC50 – Ф2). However, IC50s and EC50s of ZnO–NPs were smaller than those of Zn2+ (from ZnO–NPs ultrafiltrate and ZnCl2), indicating that dissolved Zn2+ can only partially explain the toxicity of ZnO–NPs. SEM images showed that ZnO–NPs attached on the diatom frustule surface, suggesting that the interaction between the nanoparticles and the cell surface may acerbate the toxicity of ZnO–NPs. Our results linked the physicochemical characteristics of ZnO–NPs in seawater with their toxicities to the marine diatom and highlighted the importance of salinity as an influential environmental factor governing the aggregation, dissolution and the toxicity of ZnO–NPs.

Effect of amino acid substitution in the staphylococcal peptides warnericin RK and PSMα on their anti-Legionella and hemolytic activities.

Warnericin RK from Staphylococcus warneri and PSMα from Staphylococcus epidermidis are anti-Legionella peptides which were differently classified in a previous study according to their mode of action. Indeed, warnericin RK is highly hemolytic with a bactericidal mode of action, whereas PSMα is poorly hemolytic with a bacteriostatic mode of action toward L. pneumophila. In order to find anti-Legionella peptides which are not hemolytic, a collection of peptides varying in sequence from warnericin RK to PSMα were designed and synthesized, and their anti-Legionella activities, in terms of growth inhibition, permeabilization, and bactericidal effect, as well as their hemolytic activities, were measured and compared. The results showed that some residues, at position 14 for both peptides for instance, were of major importance for bactericidal and hemolytic activities.

Abstract 747: Evaluation of Src as a therapeutic target and development of biomarkers of Src inhibitor in cancer

Abstract Background: Src is a nonreceptor tyrosine kinase involved in the crosstalk and mediation of many signaling pathways that promote cell proliferation, invasion and angiogenesis. Elevation of Src activity has been reported in many types of cancers including gastric cancer (GC) and biliary tract cancer (BTC). The purpose of this study is to evaluate Src as a therapeutic target and to elaborate the biomarkers of Src inhibitor in GC and BTC. Methods: Ten gastric cancer cell lines (SNU-1, 5, 16, 216, 601, 620, 638, 668, 719, NCI-N87) and 8 biliary tract cancer cell lines (SNU-245, 308, 478, 869, 1079, 1196, HuCCT1, TFK1) were used. Saracatinib and bosutinib were used as Src inhibitors. MTT assay, colony formation assay and 3D culture were done for determining growth inhibitory effect of Src inhibitors, alone or in combination with chemotherapeutic agents (5-FU, gemcitabine, cisplatin). Cell cycle analysis was done by FACS Calibur flow cytometer. Matrigel invasion assay and wound healing assay were done. The methods described by Chou and Talalay were used to determine whether a synergistic effect existed between drug combination. Tumor xenograft model was made and used for in vivo test of Src inhibitors. Results: Among 10 GC cells, SNU216 and NCI-N87 were sensitive to Src inhibitor. These sensitive cells showed high levels of pSRC(Y416) and pFAK (Y861, Y397, Y925). These 2 sensitive GC cells are both HER2 amplified cells. However, HER2-positive breast cancer (BC) cells (SKBR3, BT474, MDA-MB453) were resistant to Src inhibitor. Contrast to these resistant BC cells, SNU216 and N87 showed high expression of integrin αV, β4 and β8. Especially, in case of integrin β8, the mRNA/protein levels were highest in SNU216 and N87 among all GC cells. Src inhibitor-sensitive GC cells showed the apoptosis by Src inhibitor and synergism with 5-FU in vitro and in vivo. Among 8 BTC cells, 3 cells were sensitive to Src inhibitor (SNU308, SNU478 and HuCCT1) in terms of growth inhibition and migration/invasion inhibition. Sensitive cells showed high levels of integrin α2, α3 and β4. Src inhibitor induced G1 arrest and decreased pSrc, pFAK, and pERK in sensitive cells. Inhibition of pSrc was accompanied with increase of PTEN and decrease of pAKT. Src inhibitor showed the synergistic effects with cytotoxic chemotherapeutic agents (gemcitabine and cisplatin) in vitro and in vivo. When the Src was inhibited, pSTAT3 was increased thru increase of IL-6 in some cells. Conclusion: Taken together, Src could be a potential therapeutic target in gastric cancer and biliary tract cancer. The role of integrin as a biomarker for Src inhibitor should be further investigated. Citation Format: Ah-Rong Nam, Hyun-Jin Nam, Kyo Hwa Kang, Ji Eun Park, Tae Yong Kim, Sae-Won Han, Sang-Hyun Song, Seock-Ah Im, Tae-You Kim, Do-Youn Oh, Yung-Jue Bang. Evaluation of Src as a therapeutic target and development of biomarkers of Src inhibitor in 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 747. doi:10.1158/1538-7445.AM2014-747

English

The antagonistic activity against Fusarium oxysporum f. sp. ciceris was determined for 40 chickpea rhizobacteria. Twenty eight isolates showed antagonistic activity against test fungus ranging from 18.2 to 41.8%. Characterization of the antagonistic attributes showed that all the antagonistic isolates produced diffusible and volatile antifungal metabolites in terms of growth inhibition, maximum being with the isolates 39P (77.8%) and 15B (64.2%), respectively. Nineteen of the isolates showed catechol and hydroxamate type siderophore production. All the isolates produced ammonia and twelve showed HCN production. On the basis of their antagonistic and PGP functionality traits, five isolates (2B, 7B, 28P, 34P and 38P) were selected for glass house studies on two chickpea varieties (JG-62 and GPF-2). Isolates 28P, 34P and 38P were found to be most promising for wilt control and plant growth promotion. Isolate 38P reduced the wilt incidence to 44.6% which was at par with fungicide treatment (55.5%) and had a significant edge over negative control (85%) in the chickpea variety JG-62. Similar trend of wilt incidence was observed in GPF-2 variety. Green house experiments on two varieties of chickpea JG-62 and GPF-2 showed that seed treatment with plant growth promoting rhizobacteria (PGPR) + Mesorhizobia had a synergistic effect in terms of disease control and growth promotion as compared to use of single bioinoculants, thus positively influencing plant microbe interaction.   Key words: Antagonism, chickpea, Fusarium, plant growth promoting rhizobacteria (PGPR), Rhizobium.

The Impact of Chemical Compounds on Benthic Invertebrates from the Danube and Danube Delta Systems

The aim of this study is to assess freshwater sediment in terms of biological and preliminary toxicological control linked to a series of variables along the Danube River and Danube Delta systems. The research is focused on eight points of the Romanian sector in the summer to autumn of 2012. Results show a high concentration of metals, pesticides, and petroleum products in sediment samples in the monitored period. The survey is designed to gauge the total chemical pollution effects on the composition, living, and growth of benthic organisms. To assess the toxic hazard of contaminated sediment samples, a microbiotest with meiobenthic ostracods Heterocypris incongruens was performed with evaluation of mortality and growth-inhibition percentages. The preliminary results showed an increase of sediment toxicity, in terms of growth inhibition (>80%), especially at Murighiol and Ivancea. The chemical pressures alongside the temporal conditions were important variables which affected benthic communities. Low benthic diversity was found in the Isaccea–St. Gheorghe Branch sector of the Danube River. In terms of ecological status, the study reveals that the Danube and Danube Delta ecosystem are eutrophic systems equilibrated for good ecological status.

Chemometrics applied to the analysis of induced phytochelatins in Hordeum vulgare plants stressed with various toxic non-essential metals and metalloids

Hordeum vulgare plants were stressed with Hg, Cd and As and their phytotoxicity was evaluated in terms of growth inhibition and total metal uptake by the plant. The synthesised phytochelatins ((γ-Glu-Cys)n-Gly, n=2-5; PCs) were determined by HPLC with amperometric detection at a glassy carbon electrode. The results indicate that H. vulgare is a good phytostabilisation plant due to its capacity to accumulate heavy metals in roots. Cd and Hg are the most uptake toxic elements, being Cd the most potent inducer of PCs. The data obtained on the different PCs and related peptides induced by each heavy metal were used to perform a Principal Component Analysis (PCA) of the results as a function of the contaminating toxic element or its concentration level. The nature of the stressor element could be predicted from the pattern of PCs and related peptides identified by PCA. PCs were the most strongly induced peptides under Cd and Hg stress, whereas As only tended to synthesise small thiols such as glutathione and γ-glutamylcysteine, both precursors of PCs synthesis. This finding indicates that PCs are induced at different rates depending on the metal stressor used.

Abstract B068: Inhibition of GSK-3: A novel therapeutic approach for breast cancer treatment

Abstract NF-kappaB is known to regulate human breast cancer progression, metastasis and chemoresistance. Glycogen Synthase Kinase-3beta (GSK-3beta), a serine/threonine protein kinase, is an essential positive regulator of NF-kappaB transcriptional activity and thereby GSK-3beta may represent a potential therapeutic target in human breast cancer. Our objectives were to determine the expression pattern of GSK-3beta in human breast cancer, to assess the effect of GSK-3 inhibition on breast cancer cell proliferation, survival and chemoresistance, and to evaluate our lead GSK-3 inhibitors alone and in combination with chemotherapeutic drugs for the targeted therapy of breast cancer. Using nuclear/cytosolic fractionation, Western immunoblotting and immunohistochemical staining, we observed aberrant nuclear accumulation of GSK-3beta in five breast cancer cell lines and in 7 of 8 primary human breast carcinomas and corresponding lymph node metastases whereas no detectable expression of GSK-3beta was found in benign breast tissue. Pharmacological inhibition of GSK-3 led to a decrease in proliferation and survival of breast cancer cells. Our in vitro comparison studies demonstrated that our lead GSK-3 inhibitor 9-ING-41 was significantly (30 times) more effective than GSK-3 inhibitor LY2090314 (Eli Lilly), currently in mid-stage clinical trials, in terms of growth inhibition of breast cancer cells. Inhibition of GSK-3 resulted in decreased expression of the NF-kappaB target genes Bcl-2 and XIAP and induction of apoptosis. Moreover, our lead GSK-3 inhibitor 9-ING-41 and doxorubicin acted synergistically to suppress growth of breast cancer cells. Our preliminary in vivo experiments demonstrated that monotherapy with our lead GSK-3 inhibitor 9-ING-41 (20 mg/kg) for 48 hours inhibited proliferation (as shown by BrDU assay) and induced apoptosis (as shown by TUNEL assay) in MDA-MB-468 orthotopic xenograft tumors. Moreover, in vivo toxicology study demonstrated that intravenous injections of 9-ING-41 did not adversely affect mouse complete blood counts or cause overt pathological changes in mouse organs. Our results identify GSK-3beta as a new potential drug target in breast cancer and suggest that inhibition of GSK-3 is a novel therapeutic approach to overcome NF-kappaB-mediated chemoresistance in breast cancer. Citation Format: Andrey Ugolkov, Irina Gaisina, Dmitry Malin, Peter Kulesza, Alan Kozikowski, Vincent Cryns, Andrew Mazar. Inhibition of GSK-3: A novel therapeutic approach for breast cancer treatment. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B068.

A Phenylalanine to Serine Substitution within an O-Protein Mannosyltransferase Led to Strong Resistance to PMT-Inhibitors in Pichia pastoris

Protein O-mannosyltransferases (PMTs) catalyze the initial reaction of protein O-mannosylation by transferring the first mannose unit onto serine and threonine residues of a nascent polypeptide being synthesized in the endoplasmic reticulum (ER). The PMTs are well conserved in eukaryotic organisms, and in vivo defects of these enzymes result in cell death in yeast and congenital diseases in humans. A group of rhodanine-3-acetic acid derivatives (PMTi) specifically inhibits PMT activity both in vitro and in vivo. As such, these chemical compounds have been effectively used to minimize the extent of O-mannosylation on heterologously produced proteins from different yeast expression hosts. However, very little is known about how these PMT-inhibitors interact with the PMT enzyme, or what structural features of the PMTs are required for inhibitor-protein interactions. To better understand the inhibitor-enzyme interactions, and to gain potential insights for developing more effective PMT-inhibitors, we isolated PMTi-resistant mutants in Pichia pastoris. In this study, we report the identification and characterization of a point mutation within the PpPMT2 gene. We demonstrate that this F664S point mutation resulted in a near complete loss of PMTi sensitivity, both in terms of growth-inhibition and reduction in O-mannosylglycan site occupancy. Our results provide genetic evidence demonstrating that the F664 residue plays a critical role in mediating the inhibitory effects of these PMTi compounds. Our data also indicate that the main target of these PMT-inhibitors in P. pastoris is Pmt2p, and that the F664 residue most likely interacts directly with the PMTi-compounds.

Celastrol exerts synergistic effects with PHA-665752 and inhibits tumor growth of c-Met-deficient hepatocellular carcinoma in vivo

PHA665752 (PHA), a selective small molecule c-Met Inhibitor, potently inhibited HGF-stimulated and constitutive c-Met phosphorylation, as well as HGF and c-Met-driven phenotypes of a variety of tumor cells including hepatocellular carcinoma cells. However, these effects were impaired in c-Met-deficient cancer cells. In the present study, we investigated the potential anti-human c-Met-deficient hepatocellular carcinoma effects of Celastrol, a novel triterpene, and its combination with PHA. Human hepatocellular carcinoma cells BEL-7402 (c-Met-positive) and Huh7 (c-Met-deficient) were treated with different dose of PHA with or without equal dose of Celastrol, and cell growth, cell cycle and apoptosis were evaluated, respectively, by MTT assay, flow cytometry and Caspase3/7 activity. Nude mice bearing Huh7 xenografts were used to assess the in vivo anti-tumor activity. Our results showed that Celastrol at high concentration (>1.0 μM) induced G2/M arrest and apoptosis with the activation of Caspase3/7 in Huh7 cells whereas at low concentration (<1.0 μM) had no obvious effects. Low concentration Celastrol presented significant combined effects with PHA on Huh7 cells and Huh7 xenografts in terms of growth inhibition, migration inhibition and apoptosis induction. These results suggest that Celastrol and its combination with PHA present the therapeutic potential on c-Met-deficient hepatocellular carcinoma, and deserve further preclinical and clinical studies.