Mechanism Of Growth Inhibition Research Articles

Magnetic nanoparticle-mediated hyperthermia therapy induces tumour growth inhibition by apoptosis and Hsp90/AKT modulation

Purpose: We have evaluated the hyperthermia efficacy of oleic acid-functionalised Fe3O4 magnetic nanoparticles (MN-OA) under in vivo conditions and elucidated the underlying mechanism of tumour growth inhibition. Materials and methods: The efficacy and mechanism of tumour growth inhibition by MN-OA-mediated magnetic hyperthermia therapy (MHT) was evaluated in a murine fibrosarcoma tumour model (WEHI-164) using techniques such as TUNEL assay, Western blotting (WB), immunofluorescence (IF) staining and histopathological examination. In addition, bio-distribution of MN-OA in tumour/other target organs and its effect on normal organ function were studied by Prussian blue staining and serum biochemical analysis, respectively. Results: MN-OA-induced MHT resulted in significant inhibition of tumour growth as determined by measurement of tumour volume, as well as by in vivo imaging of tumour derived from luciferase-transfected WEHI-164 cells. Histopathology analysis showed presence of severe apoptosis and reduced tumour cells proliferation, which was further confirmed by TUNEL assay, reduced expression of Ki-67 and enhanced level of cleaved caspase-3, in tumours treated with MHT. Moreover, expression of heat stress marker, Hsp90 and its client protein, AKT/PKB was reduced by ∼50 and 80%, respectively, in tumours treated with MHT as studied by WB and IF staining. Serum analysis suggested insignificant toxicity of MN-OA (in terms of liver and kidney function), which was further correlated with minimal accumulation of MN-OA in target organs. Conclusions: These results suggest the involvement of apoptosis and Hsp90/AKT modulation in MN-OA-mediated MHT-induced tumour growth inhibition.

Efficacy of Mentha spicata essential oil in suppression of Aspergillus flavus and aflatoxin contamination in chickpea with particular emphasis to mode of antifungal action.

The present study reports in vivo antifungal and antiaflatoxigenic efficacy of Mentha spicata essential oil (EO) against toxigenic Aspergillus flavus strain LHP(C)-D6 in chickpea food system up to 12 months of storage. In addition, the mode of antifungal action of EO was also determined to understand the mechanism of fungal growth inhibition. The in vivo study with different concentrations of M. spicata EO showed dose-dependent decrease in fungal colony count as well as aflatoxin B1 concentration. The EO caused >50% protection in inoculated sets and >70% protection in uninoculated sets of chickpea food system against A. flavus at 1.0 μL mL(-1) air concentration. However, at the same concentration, EO caused 100% inhibition to aflatoxin B1 production in both sets when analyzed through high-performance liquid chromatography (HPLC). The antifungal target of EO in fumigated cells of A. flavus was found to be the plasma membrane when analyzed through electron microscopic observations and ions leakage test. The EO fumigated chickpea seeds showed 100% seed germination and seedling growth after 12 months of storage. Based on these observations, M. spicata EO can be recommended as plant-based preservative for safe protection of food commodities during storage conditions against fungal and most importantly mycotoxin contaminations.

Anti-leukemia efficacy and mechanisms of action of SL-101, a novel anti-CD123 antibody-conjugate in acute myeloid leukemia

Outcomes of acute myeloid leukemia (AML) remain poor and warrant the development of novel therapeutic agents. CD123 (interleukin-3 [IL-3] receptor alpha subunit), is overexpressed on AML blasts and leukemia progenitor/stem cells (LSCs) compared to normal hematopoietic cells (Jordan et al. Leukemia 2000). SL-101 is a novel anti-CD123 antibody-conjugate comprised of anti-CD123 scFv fused to a truncated and optimized pseudomonas exotoxin (PE) lacking its native targeting domain. We have previously demonstrated SL-1019s cell killing efficacy in AML cell lines (Han et al. ASH 2013). Here we report the anti-tumor efficacy of SL-101 against primary AML cells and the underlying mechanisms of its cytotoxicity. Fourteen genetically diverse primary AML samples were treated with various doses of SL-101 for 48 h. Most samples express high levels of CD123 (median 89.9%, range 20.4-99.3%) and intermediate levels of CD131 (median 54.0%, range 9.0-91.1%), the IL-3 beta subunit. SL-101 was highly active against AML samples, with an IC50 of 0.19 µg/ml (range 0.003 - 0.98 µg/ml). No significant correlation was found between SL-101 activity and levels of CD123 or CD131 (n=12). SL-101 also selectively and significantly suppressed AML colony formation (69.5% ± 15.0% inhibition of total colonies, n=7) while sparing normal bone marrow (5.6% ± 3.3% inhibition, n=4; p=0.0001) (Fig. 1A). We next investigated the mechanisms of the cytotoxic activity of SL-101. Using annexinV/DAPI flow cytometry, we first evaluated the induction of apoptosis in AML blasts and phenotypically defined CD45dimCD34+CD38-CD123+ LSCs. After 48 h treatment, SL-101 at 1.0 µg/ml induced higher specific apoptosis in the AML LSCs (51.2% ± 25.4%) than in blasts (39.4% ± 19.0%, p=0.006, n=10). Quantification of the annexinV–/DAPI– viable cells using counting beads demonstrated further reduction of cell numbers by SL-101 (LSCs 72%, blasts 64.6%), indicating additional mechanisms of cell growth inhibition. It was recently demonstrated that upon internalization, PE traffics through the endoplasmic reticulum to the cytosol, where it inactivates protein synthesis by catalyzing ADP ribosylation of elongation factor 2 and causes non-apoptotic cell death (Wayne et al. Blood 2014). To examine the contribution of the direct inhibition of protein synthesis by PE, we first studied SL-101 internalization utilizing DyLight 680-labeled SL-101 by flow cytometry and fluorescence imaging. In CD123-expressing AML cell lines MV4-11 and MOLM13, the intracellular median intensity of DyLight 680 signal increased 5.4- and 3.5-fold, respectively, within 4 h of treatment and 22.3- and 16.3-fold after 24 h (Fig 1B). Fluorescence imaging confirmed cytosolic localization of SL-101 in both cell lines, demonstrating efficient cellular uptake of SL-101. We also examined the efficacy of SL-101 in inhibiting nascent protein synthesis in MV4-11 cells using an AHA Alexa Fluor 488 protein synthesis assay. SL-101 significantly reduced protein synthesis (40.3%, p=0.0005) within 4 h (Fig 1C), even at low concentrations (0.01 µg/ml), which was comparable to the positive control cycloheximide (44.8% ± 7.9%, p=0.0001). These findings confirmed the potential of SL-101 to efficiently internalize and promote cell death through protein synthesis blockade. We further investigated the ability of SL-101 to inhibit intracellular signaling in response to IL-3. To this end, cytokine-dependent Mo7e leukemia cells were serum starved and pre-treated with SL-101 at 1.0 µg/ml overnight, followed by stimulation with IL-3. SL-101 significantly suppressed IL-3-induced activation of p-STAT5 (57.1% ± 2.6% inhibition, p=0.003) and modestly inhibited p-AKT (17.4% ± 5.4% inhibition, p=0.04), but not p-ERK signaling (Fig. 1D). In summary, our data demonstrate that the novel anti-CD123 antibody-conjugate, SL-101, is highly active in AML and induces growth arrest and apoptosis in AML blasts and LSCs by inhibiting protein synthesis and interfering with IL-3 signal transduction pathways. Ongoing studies that will be reported at this meeting investigate the in vivo anti-leukemia efficacy of SL-101 in NSG mice engrafted with primary AML cells. In conclusion, SL-101 is a novel, potent antibody-conjugate directed against AML blasts and LSCs, and our studies warrant further development of this agent. Disclosures Rowinsky:Stemline Therapeutics: Employment, Equity Ownership. Brooks:Stemline Therapeutics: Employment, Equity Ownership.

Phytotoxic mechanisms of bur cucumber seed extracts on lettuce with special reference to analysis of chloroplast proteins, phytohormones, and nutritional elements

Bioherbicides from plant extracts are an effective and environmentally friendly method to prevent weed growth. The present investigation was aimed at determining the inhibitory effect of bur cucumber seed extracts (BSE) on lettuce plant growth. Bur cucumber seeds were ground with water, and two different concentrations of seed extracts (10% and 20%) were prepared and applied to lettuce plants. Decreased plant height, number of leaves, leaf length, leaf width, anProd. Type: FTPd leaf area were found in lettuce exposed to BSE as compared with controls. A significant reduction in lettuce biomass was observed in 20% BSE-treated plants due to the presence of higher amounts of phenolic content in the extracts. Moreover, a significant inhibitory chemical, 2-linoleoyl glycerol, was identified in BSE extracts. The mechanism of plant growth inhibition was assayed in lettuce proteins by 2-dimensional electrophoresis (2-DE) and the LC-MS/MS method. In total, 57 protein spots were detected in plants treated with 20% BSE and control plants. Among these, 39 proteins were down-regulated and 18 proteins were up-regulated in plants exposed to 20% BSE as compared with controls. The presence of low levels of chlorophyll a/b binding protein and oxygen-evolving enhancer protein 1 in BSE-exposed plants reduced photosynthetic pigment synthesis and might be a reason for stunted plant growth. Indeed, the plant-growth stimulating hormone gibberellin was inhibited, and synthesis of stress hormones such as abscisic acid, jasmonic acid, and salicylic acid were triggered in lettuce by the effects of BSE. Uptake of essential nutrients, Ca, Fe, Mg, K, S, and Mo, was deficient and accumulation of the toxic ions Cu, Zn, and Na was higher in BSE-treated plants. The results of this study suggest that extracts of bur cucumber seeds can be an effective eco-friendly bioherbicide for weed control that work by inhibiting mechanisms of photosynthesis and regulating phytohormones and nutritional elements.

Disruption of pre-B-cell receptor signaling jams the WNT/β-catenin pathway and induces cell death in B-cell acute lymphoblastic leukemia cell lines

Targeting components of the B-cell receptor (BCR) pathway have dramatically improved clinical outcomes in a variety of B-cell malignancies. Despite the well-documented pathogenic role of BCR precursor (pre-BCR) pathway in B-cell acute lymphoblastic leukemia (B-ALL), there is limited available data of therapies that aim to disrupt this pathway. To investigate the role of protein kinase Cβ (PKCβ), a crucial mediator of BCR and pre-BCR signaling, in B-ALL survival, we studied the activity of the PKCβ selective inhibitor enzastaurin (ENZ) in seven B-ALL cell lines. Treatment with ENZ resulted in a dose- and time-dependent growth inhibition in all cell lines with a relatively higher efficacy in pro-B ALL with translocation t(4;11)(q21;q23). The mechanism of growth inhibition was by apoptotic induction and cell cycle arrest. A rapid reduction in phosphorylation of AKT and its downstream target glycogen synthase kinase 3β (GSK3β) were observed at 30min after treatment and remaining for 48h. The reduction in GSK3β phosphorylation was associated with a paradoxical accumulation of β-catenin, which was due to a transient loss of β-catenin phosphorylation at ser33-37. In addition, accumulation of β-catenin was associated with downregulation of c-Myc, upregulatiuon of c-Jun, and a subsequent protective effect on the tumor suppressor p73.Data in this paper were presented in part at 2012 American Society of Hematology Annual Meeting, abstract 1350.

Abstract 469: mPGES1 deletion and the mechanisms of tumor growth suppression

Abstract The pro-tumor contributions of prostaglandin (PG) E2 are established, as is the clinical efficacy of pharmacological inhibition of COX-2, the rate-limiting enzyme in PGE2 synthesis. In addition to the desired suppression of tumor PGE2, collateral loss of endothelial COX-2-derived PGI2 imposes a cardiovascular hazard that limits clinical use of COX-2 inhibitors. mPGES1, the terminal enzyme in the PGE2 synthesis pathway, is an alternative target to interrupt COX-2-driven events in tumors without elevating cardiovascular risk. We engineered mice transgenic for an activated HER2/neu oncogene to lack mPGES1 globally (mPGES1 KOgl) or only in mammary epithelial cells (mPGES1 KOepi). Abdominal mammary glands from wild type (WT), heterozygous (het), mPGES1 KOgl, and mPGES1 KOepi mice were harvested at 22 weeks of age, paraffin embedded, H&E stained and scanned for whole slide imaging (WSI). Tumor multiplicity was calculated based on the number of lesions on WSI. Adjacent sections were immunostained for Ki67, caspase 3, Factor VIII, CD3, and F4/80, to assess proliferation, apoptosis, angiogenesis, and tumor lymphocyte and macrophage infiltration. Tumor multiplicity was significantly higher in WT compared to either mPGES1 KOgl or mPGES1 KOepi mice. Concordantly, shRNA knock down (KD) of mPGES1 in mammary tumor cells dramatically suppressed their growth as orthotopic tumors in syngeneic immune competent WT hosts: 4 weeks after the injections, 6 out of 6 non-target shRNA control tumors grew successfully while 6 out of 10 mPGES1 KD cells failed to grow. In separate experiments, 14 of 14 non-target shRNA cells formed tumors after 2 weeks while 12 out of 18 mPGES1 KD tumors failed. By immunohistochemistry, no difference was observed in proliferation and apoptosis markers, as well as in the number of tumor infiltrating macrophages and lymphocytes, between WT, mPGES1 KOgl, and mPGES1 KOepi tumors. In contrast to our previous study in mice lacking epithelial COX-2 (COX-2 KOepi), vascularization was not different between mPEGS-1 KOepi and WT tumors suggesting divergence in the anti-tumorigenic mechanisms in mPEGS-1 KOepi and COX-2 KOepi tumors. By flow cytometry there was no difference in infiltrating macrophage and lymphocyte density or functional phenotypes, between WT, mPGES1 KOepi, and mPGES1 KD orthotopic tumors. Similarly, no difference was observed in the number of macrophages and myeloid derived suppressor cells in spleens from mPGES1 sufficient and mPGES1 KD orthotopic tumor bearing mice. In contrast, decreased co-inhibitory molecule PD-1 expressing CD4+ and CD8+ lymphocyte numbers were evident in draining lymph nodes in mice injected with mPGES1 KD cells compared to WT. This finding indicates that the absence of mPGES1-derived PGE2 renders tumor cells less efficient in creating and maintaining an immunosuppressive tumor microenvironment and that targeting mPGES1 may enhance anti-tumor immune mechanisms. Citation Format: Nune Markosyan, Andrew Rech, Robert H. Vonderheide, Garret A. FitzGerald, Emer M. Smyth. mPGES1 deletion and the mechanisms of tumor growth suppression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 469. doi:10.1158/1538-7445.AM2015-469

Abstract 1638: Preclinical characterization of the novel TTK kinase inhibitor S81694 for the treatment of triple negative breast cancer

Abstract The tyrosine threonine kinase TTK, also known as Monopolar Spindle 1 kinase (MPS1), is a conserved kinase found to be highly expressed in a number of human tumors of different origin. TTK kinase activity plays a critical role in the control of mitosis, regulating the spindle assembly checkpoint (SAC), a mitotic mechanism required for proper chromosome alignment and segregation during cellular division. The activity of this checkpoint has been shown to be proficient and up-regulated in aneuploid tumors, a common feature measured in approximately 90% of solid tumors and 70% of hematological cancers. S81694 (NMS-P153) is a novel potent inhibitor of TTK. It is a tight binder compound with long residence time on the target and is highly selective for TTK against a wide range of tested enzymes (kinases, channels, hormones, GPCRs). In vitro, brief exposure is sufficient to commit cells to death with the compound showing anti-proliferative activity with a demonstrated mechanism of action and marked tumor growth inhibition in preclinical in vivo tumor models associated with a good pharmacokinetic profile in both rodent and non-rodent species. TTK inhibitors are anticipated to be more active in SAC dependent tumors and data in the literature suggests that triple negative breast cancer is among the tumor types potentially highly dependent on TTK activity. Moreover, in accelerating mitosis, TTK inhibitors have a novel mode of action as compared to currently known mitotic inhibitors. To confirm the role of TTK in breast cancer and identify a sensitive patient population to S81694 treatment, translational studies have been conducted on a large panel of human tumor cell lines, including 39 of mammary gland origin. Anti-proliferative activity was measured after 72h continuous treatment or after a brief treatment of 7h followed by 2D colony forming assay to verify the long term effects of a treatment which mimics compound half-life in mice. In addition, active caspase 3 induction was also analyzed to identify cell lines more prone to activate apoptosis upon treatment. Triple negative breast cancer (TNBC) cell lines were confirmed to be particularly sensitive to S81694 and correlation was observed between sensitivity profile in vitro and tumor growth inhibition in vivo. In MDA-MB-231 xenograft and orthotopic models, tumor regression, cured animals and metastasis reduction in several organs including brain and lungs was observed. These results further confirm the role of TTK in triple negative breast cancer and could support the clinical development of S81694 in this indication. Citation Format: Riccardo Colombo, Mike Burbridge, Marianne Rodriguez, Frédérique Cantero, Marina Caldarelli, Maria Laura Giorgini, Francesco Sola, Dario Ballinari, Marina Ciomei, Roberta Bosotti, Alessia Montagnoli, Antonella Isacchi, Daniele Donati, Arturo Galvani. Preclinical characterization of the novel TTK kinase inhibitor S81694 for the treatment of triple negative breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1638. doi:10.1158/1538-7445.AM2015-1638

Abstract 1914: A novel celecoxib derivative that lacks COX-2 inhibition but displays potent colon tumor cell growth and PDE5 inhibitory activity

Abstract Background: The use of celecoxib for cancer chemoprevention is not recommended because of modest efficacy and potentially fatal cardiovascular toxicity due to COX-2 inhibition. There is evidence that celecoxib inhibits tumor growth by a COX-2 independent mechanism, suggesting that efficacy and safety could be improved by chemical optimization towards the underlying target. We synthesized a novel series of celecoxib derivatives that lack COX-2 inhibitory activity, yet display higher potency to inhibit colon tumor cell growth. Here we study a potential mechanism involving phosphodiesterase 5 (PDE5) inhibition, elevation of intracellular cGMP levels, PKG activation and suppression of β-catenin signaling. Methods: The IMAP fluorescence polarization PDE assay was used to measure PDE activity. Colon tumor cell lines (HT-29 and HCT-116) were used to measure growth inhibitory activity using the Cell Titer Glo Assay. COX inhibitory activity was measured using a fluorescent assay. Intracellular cGMP levels in live cells were measured using a cGMP biosensor assay, while cGMP levels in cell and tissues lysates were measured by EIA assay. PKG activity was measured by western blotting using a phosphospecific antibody to VASP. Levels of β-catenin, survivin, and cyclin D1 were determined by western blotting. TCF/LEF transcriptional activity was measured using a luciferase reporter assay. Cell cycle arrest was measured using an IXM imager and MetaXpress software. Oral bioavailability was determined based on drug concentrations in plasma and colon mucosal tissue after treatment by oral gavage in C57BL/6 mice. Results: Celecoxib inhibits HT-29 and HCT-115 colon tumor cell growth and PDE5 activity with IC50 values of 34.3, 35.3, and 37.0 μM, while a derivative, RF26, had values of 5.8, 6.6, and 0.001 μM, respectively. RF26 was highly selective for PDE5 compared with other PDE isozymes. Celecoxib displays an IC50 of 0.058 μM for COX-2 inhibition, while RF26 lacks COX-2 inhibitory activity. RF26 increases cGMP levels, activates PKG, and decreases β-catenin levels and activity demonstrated by decreased survivin and cyclin D1 protein levels and TCF/LEF transcriptional activity. The mechanism of growth inhibition involves G1 cell cycle arrest. In mice, RF26 displays promising pharmacokinetics and drug levels in the target tissue that increase cGMP levels. Conclusions: A novel series of celecoxib derivatives was identified that lack COX-2 inhibitory activity; yet display high potency to inhibit colon tumor cell growth. PDE5 inhibition appears to be an important mechanism for this class of compounds, which is consistent with previous observations from our group implicating cGMP/PKG activation as an anticancer target. These findings may lead to the development of novel celecoxib derivatives with improved anti-tumor efficacy and reduced risk of toxicity. Supported by NIH grants 1R01CA155638 and 1R01CA131378 (Piazza). Citation Format: Sara C. Sigler, Veronica Ramirez-Alcantara, Adam B. Keeton, Mohammad Abdel-Halim, Ashraf H. Abadi, Gary A. Piazza. A novel celecoxib derivative that lacks COX-2 inhibition but displays potent colon tumor cell growth and PDE5 inhibitory activity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1914. doi:10.1158/1538-7445.AM2015-1914

Design, synthesis and biological evaluation of 1,3-diphenyl-1H-pyrazole derivatives containing benzimidazole skeleton as potential anticancer and apoptosis inducing agents

A series of forty different pyrazole containing benzimidazole hybrids (6–45) have been designed, synthesized and evaluated for their potential anti-proliferative activity against three human tumor cell lines - lung (A549), breast (MCF-7), and cervical (HeLa). Some of the compounds, specifically 9, 17, and 28, showed potent growth inhibition against all the cell lines tested, with IC50 values in the range of 0.83–1.81 μM. Breast cancer cells were used for further detailed studies to understand the mechanism of cell growth inhibition and apoptosis inducing effect of compounds. The morphology, cell migration and long term clonogenic survival of MCF-7 breast cancer cells were severely affected by treatment with these compounds. Flow-cytometry revealed the compounds arrested MCF-7 cells in the G1 phase of the cell cycle via down regulation of cyclin D2 and CDK2. Fluorescent staining and DNA fragmentation studies showed that cell proliferation was inhibited by induction of apoptosis. Moreover, the compounds led to collapse of mitochondrial membrane potential (DΨm) and increased levels of reactive oxygen species (ROS) were noted. The ease of synthesis and the remarkable biological activities make these compounds promising new frameworks for the development of cancer therapeutics.

Hydroxyapatite Growth Inhibition Effect of Pellicle Statherin Peptides

In our recent studies, we have shown that in vivo–acquired enamel pellicle is a sophisticated biological structure containing a significant portion of naturally occurring salivary peptides. From a functional aspect, the identification of peptides in the acquired enamel pellicle is of interest because many salivary proteins exhibit functional domains that maintain the activities of the native protein. Among the in vivo–acquired enamel pellicle peptides that have been newly identified, 5 peptides are derived from statherin. Here, we assessed the ability of these statherin pellicle peptides to inhibit hydroxyapatite crystal growth. In addition, atomistic molecular dynamics (MD) simulations were performed to better understand the underlying physical mechanisms of hydroxyapatite growth inhibition. A microplate colorimetric assay was used to quantify hydroxyapatite growth. Statherin protein, 5 statherin-derived peptides, and a peptide lacking phosphate at residues 2 and 3 were analyzed. Statherin peptide phosphorylated on residues 2 and 3 indicated a significant inhibitory effect when compared with the 5 other peptides (P < 0.05). MD simulations showed a strong affinity and fast adsorption to hydroxyapatite for phosphopeptides, whereas unphosphorylated peptides interacted weakly with the hydroxyapatite. Our data suggest that the presence of a covalently linked phosphate group (at residues 2 and 3) in statherin peptides modulates the effect of hydroxyapatite growth inhibition. This study provides a mechanism to account for the composition and function of acquired enamel pellicle statherin peptides that will contribute as a base for the development of biologically stable and functional synthetic peptides for therapeutic use against dental caries and/or periodontal disease.

YAPing Hippo Forecasts a New Target for Lung Cancer Prevention and Treatment.

YAPing Hippo Forecasts a New Target for Lung Cancer Prevention and Treatment.

Mechanisms of environmental chemicals that enable the cancer hallmark of evasion of growth suppression.

As part of the Halifax Project, this review brings attention to the potential effects of environmental chemicals on important molecular and cellular regulators of the cancer hallmark of evading growth suppression. Specifically, we review the mechanisms by which cancer cells escape the growth-inhibitory signals of p53, retinoblastoma protein, transforming growth factor-beta, gap junctions and contact inhibition. We discuss the effects of selected environmental chemicals on these mechanisms of growth inhibition and cross-reference the effects of these chemicals in other classical cancer hallmarks.

Bioactivity of endophytic bacteria and yeasts isolated from Thymus

Endophytes are important resources of bioactive compounds and due to their potential in theproduction of secondary metabolites, they are regarded as a potential reservoir ofbiotechnological applications. In this study, the bioactivity of bacteria and yeasts endophytesresiding in Thymus sp. was evaluated. During April to October 2011, symptomless and healthytissues of Thymus sp. were collected. A total of 23 strains of endophytic bacteria and 6 yeastswere isolated. The bio-effects of the endophytes were studied on Botrytis cinerea and plantpathogenic bacteria Xanthomonas arboricola pv. juglandis and Streptomyces scabies andhuman pathogens Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC. Growthinhibition mechanisms of the endophytes against B. cinerea were evaluated and it seems that theantimicrobial effects of bacterial endophytes are related to the production of protease enzyme,hydrogen cyanide and volatile compounds. Bacterial strains were identified as Bacillus,Pseudomonas and Xanthomonas of which Bacillus was the predominant isolate. For the firsttime, Bacillus is reported from Thymus eriocalyx, T. lancifolius, T. fallax, T. kotschyanus and T.vulgaris and Pseudomonas and Xanthomonas as endophytic bacteria from Thymus.

Effect of Tin Oxide Additive on the Suppression of Dendrite Growth of Zinc Electrodeposits

Unlike the market acceptance of Zn-air primary battery, the Zn-air secondary battery is not yet commercialized, mainly due to its poor cycling stability. Although a number of issues have been raised as possible reasons for its low electrical rechargeability, the dendrite growth of Zn during charging process might be most critical because it leads to short circuits and thereby catastrophic failure. Many inorganic additives have been reported for the suppression of the dendritic growth of Zn electrodeposits. For example, it is known that a small amount of Pb ions in an alkaline solution effectively suppresses the Zn dendritic growth. Nevertheless, the environmental issues of Pb-containing additive drive the researchers to search for its alternatives. Sn is cost effective as well as environmentally friendly. Since it has higher reduction potential than Zn, it might be co-deposited with Zn to possibly change the growth habit of Zn dendrite. In fact, it has been reported that Sn ions affected the Zn deposition on a Zn single crystal at the very low Sn content, implying that Sn can be a possible option to toxic and expensive additives. In this work, we report that SnO additive significantly changes the morphology of Zn electrodeposits and suppresses quite well its dendritic growth. From the cyclic voltammograms at different SnO contents, it is proved that a few milli-moles of Sn ions in the electrolyte have a strong effect on the Sn deposition process. Moreover, the microscopic observation reveals that the non-uniform and dendritic growth of Zn electrodeposits is considerably suppressed at high content of SnO additive. From the depth profiling of the composition of Zn electrodeposit, it is confirmed that Sn is co-deposited with Zn during the course of deposition process, strongly indicating that the incorporated Sn interferes the dendritic growth of Zn. However, the addition of excess SnO resulted in a rough surface and crumbling porous particles containing large amount of Sn phase. In this presentation, an optimal SnO content required for the creation of dense Zn electrodeposits will be highlighted. Moreover, the possible mechanism of dendrite growth suppression by Sn ions will be further discussed.

Origin of p-type conduction in Cu-doped ZnO nano-films synthesized by hydrothermal method combined with post-annealing

Cu-doped ZnO nano-films were synthesized by a hydrothermal method combined with post-annealing process. The mechanism for the formation of the nano-films can be explained by a Cu-adsorbed face-selective crystal growth inhibition mechanism. The 2at.% Cu-doped sample behaved p-type conductivity, which can be verified by a p–n homojunction with well-defined rectifying characteristic. The origin of p-type conduction of the Cu-doped sample was studied by XPS and PL analyses. The CuZn+1 acceptor with the ionization energy of 326meV should be the main reason for the p-type conductivity.

Caudatin Inhibits Human Glioma Cells Growth Through Triggering DNA Damage-Mediated Cell Cycle Arrest.

Caudatin, one of the species of C-21 steroidal glycosides mainly isolated from the root of Cynanchum bungei Decne, exhibits potent anticancer activities. However, the mechanism remains poorly defined. In the present study, the growth inhibitory effect and mechanism of caudatin on human glioma cells were evaluated in vitro. The results revealed that caudatin time- and dose-dependently inhibited U251 and U87 cells growth. Flow cytometry analysis indicated that caudatin-induced growth inhibition against U251 and U87 cells was mainly achieved by the induction of G0/G1 and S-phase cell cycle arrest through triggering DNA damage, as convinced by the up-regulation of p53, p21, and histone phosphorylation, as well as the down-regulation of cyclin D1. Moreover, caudatin treatment also triggered the activation of ERK and inactivation of AKT pathway. LY294002 (an AKT inhibitor) addition enhanced caudation-induced AKT inhibition, indicating that caudatin inhibited U251 cells growth in an AKT-dependent manner. Taken together, these results indicate that caudatin may act as a novel cytostatic reagent against human glioma cells through the induction of DNA damage-mediated cell cycle arrest with the involvement of modulating MAPK and AKT pathways.

Vanadium complexes inhibit the growth of HT‐29 and MCF‐7 cancer cell lines

Cancer is characterized by an over‐proliferation of cells that are no longer within control of the cell cycle. Platinum chemotherapeutic agents have been used effectively in cancer treatment but with adverse toxic effects. Recent studies suggest that complexes using vanadium (VCs) have effectively slowed or inhibited cancer cell growth through many methods, including the activation of apoptotic pathways. We synthesized novel VCs possessing different functional groups for this study. We hypothesized that these VCs act to inhibit cancer cell growth via induction of caspase 3/7. HT‐29 and MCF‐7 cell lines were used as models of colon and breast cancer, respectively. Three different VCs were synthesized, each having a unique functional group (5‐Br, 5‐OMe, 3‐OMe) that affected the electron density distribution within the VC. Using cell proliferation assays, efficacy of each VC was assessed in vitro. Treatments were applied for 24 hours in triplicate and the experiments were repeated on three separate occasions (n=3). VO2‐Br was the most effective against both HT‐29 and MCF‐7 cell lines with 65.3% and 53.6% growth inhibition respectively. VO2‐3‐OMe yielded 46.1% and 51.5% growth inhibition of HT‐29 and MCF‐7 cells, respectively. VO2‐5‐OMe was the least effective, and yielded 13.4% growth in HT‐29 cells and 19.6% growth inhibition in MCF‐7 cells. Apoptosis assays were then performed on HT‐29 cell lines using 5‐Br to investigate possible growth inhibition mechanism. We observed no notable increase in caspase 3/7 activity of the treated groups compared to control groups. In conclusion, we found VCs to be effective at inhibiting growth of HT‐29 and MCF‐7 cell lines.

Resveratrol analogue (E)-8-acetoxy-2-[2-(3,4-diacetoxyphenyl) ethenyl]-quinazoline induces G2/M cell cycle arrest through the activation of ATM/ATR in human cervical carcinoma HeLa cells

Styrylquinazolines are synthetic analogues of resveratrol and have been suggested to cause anti-inflammatory activity by modulating prostaglandin E₂ (PGE₂) production. In the present study, we evaluated cytotoxic effects of various styrylquinazoline derivatives and found that (E)-8-acetoxy-2-[2-(3,4-diacetoxyphenyl)ethenyl]-quinazoline (8-ADEQ) most potently inhibited the proliferation of the human cervical carcinoma HeLa cells. Exploring the growth-inhibitory mechanisms of 8-ADEQ, we found that it causes a cell cycle arrest at the G₂/M phase by DNA flow cytometric analysis, which was accompanied by upregulation of cyclin B1 expression and cyclin-dependent protein kinase 1 (Cdk1) phosphorylation. In addition, we observed that 8-ADEQ causes phosphorylation of the cell division cycle 25C (Cdc25C) protein through the activation of checkpoint kinases 1 (Chk1) and Chk2, which in turn were activated via ataxia telangiectasia mutated (ATM)/ataxia telangiectasia-Rad3-related (ATR) kinases in response to the DNA damage. Furthermore, ATM/ATR inhibitor caffeine, p53- or ATM/ATR-specific siRNA significantly attenuated 8-ADEQ-induced G₂/M arrest. These results suggest that the 8-ADEQ inhibits the proliferation of human cervical cancer HeLa cells by DNA damage-mediated G₂/M cell cycle arrest. 8-ADEQ‑induced G₂/M arrest is mediated by the activation of both Chk1/2-Cdc25 and p53-p21CIP1/WAF1 via ATM/ATR pathway, and indicates that 8-ADEQ appears to have potential in the treatment of cervical cancer.

Extracellular superoxide dismutase regulates the expression of small gtpase regulatory proteins GEFs, GAPs, and GDI.

Extracellular superoxide dismutase (SOD3), which catalyzes the dismutation of superoxide anions to hydrogen peroxide at the cell membranes, regulates the cellular growth in a dose-dependent manner. This enzyme induces primary cell proliferation and immortalization at low expression levels whereas it activates cancer barrier signaling through the p53-p21 pathway at high expression levels, causing growth arrest, senescence, and apoptosis. Because previous reports suggested that the SOD3–induced reduction in the rates of cellular growth and migration also occurred in the absence of functional p53 signaling, in the current study we investigated the SOD3-induced growth-suppressive mechanisms in anaplastic thyroid cancer cells. Based on our data, the robust over-expression of SOD3 increased the level of phosphorylation of the EGFR, ERBB2, RYK, ALK, FLT3, and EPHA10 receptor tyrosine kinases with the consequent downstream activation of the SRC, FYN, YES, HCK, and LYN kinases. However, pull-down experiments focusing on the small GTPase RAS, RAC, CDC42, and RHO revealed a reduced level of growth and migration signal transduction, such as the lack of stimulation of the mitogen pathway, in the SOD3 over-expressing cells, which was confirmed by MEK1/2 and ERK1/2 Western blotting analysis. Interestingly, the mRNA expression analyses indicated that SOD3 regulated the expression of guanine nucleotide-exchange factors (RHO GEF16, RAL GEF RGL1), GTPase-activating proteins (ARFGAP ADAP2, RAS GAP RASAL1, RGS4), and a Rho guanine nucleotide-disassociation inhibitor (RHO GDI 2) in a dose dependent manner, thus controlling signaling through the small G protein GTPases. Therefore, our current data may suggest the occurrence of dose-dependent SOD3–driven control of the GTP loading of small G proteins indicating a novel growth regulatory mechanism of this enzyme.

Nostoc sp. extract induces oxidative stress-mediated root cell destruction in Mimosa pigra L.

BackgroundMimosa pigra is an invasive weed in some regions of South East Asia and Australia. Our previous study has revealed that a cyanobacterium, Nostoc sp., extract can inhibit root growth in M. pigra seedlings. In this study, some physiological processes involve oxidative stress-mediated cell death and root ultrastructure were investigated to clarify the mechanisms of root growth suppression and bioherbicidal potential of the extract.ResultsNostoc sp. extract enhanced overproduction of reactive oxygen species (ROS) at 24 h, the intensity of red fluorescence increased at 72 h, and caused a slightly increased H2O2 consistent with the activation of scavenging enzymes (catalase, ascorbic acid peroxidase, glutathione reductase, and peroxidases). This suggests that oxidative stress occurred in the presence of the extract which was supported by increased cell death and lipid peroxidation at 24 h. Reduction of malondialdehyde content and an increase in cell death at 72 h indicated oxidative damage and cellular leakage. Ultrastructural changes were determined at 72 h by scanning electron micrographs which confirmed the damage of epidermal and root cap cells and the disaggregation and destruction of root tip cells. Transmission electron micrographs showed the dissolution of the middle lamella, deposition of some substances in vacuoles, and abnormal mitochondria (swollen mitochondria and indistinct cristae).ConclusionsNostoc sp. extract enhance oxidative stress by ROS production resulting in lipid peroxidation and massive cell death despite the activation of antioxidative enzymes. Understanding mechanism of action of Nostoc sp. extract will provide information for application of the extract to use as natural herbicide for control of M. pigra.Electronic supplementary materialThe online version of this article (doi:10.1186/s40529-014-0081-3) contains supplementary material, which is available to authorized users.