Reduction In Growth Inhibition Research Articles

Extraction of phytochemicals from young shoots of Pinus sylvestris L. and analysis of their selected biological properties

Young shoots of Pinus sylvestris L. are a good source of pro-healthy substances, mainly, flavonoids and ascorbic acid. In this paper, we presented research on optimisation of ultrasound-assisted extraction of phytochemicals from young pine shoots, as well as assessment of selected biological properties of obtained extract. Research showed that the yield of antioxidants extraction from young shoots of P. sylvestris L. was dependent on the applied extraction conditions. Based on the mathematical models, the optimal conditions of extraction process in which the maximum quantity of antioxidants is achieved from the pine shoots mass unit, i.e., the ethanol concentration to 61%, and process time equal to 1201 s. Moreover, bioactive components from young pine shoots suppressed the toxic effects of cadmium in relation to Saccharomyces cerevisiae model, which was manifested by a lower ability to produce ROS, lower activity of superoxide dismutase and catalase, as well as a reduction in growth inhibition, compared to cells treated only with cadmium. In addition, extract from young pine shoots showed inhibitor activity in relation to COX-1, COX-2 and acetylcholinesterase.

Macranthoside B Induces Apoptosis and Autophagy Via Reactive Oxygen Species Accumulation in Human Ovarian Cancer A2780 Cells

ABSTRACTMacranthoside B (MB), a saponin compound in Lonicera macranthoides, can block cell proliferation and induce cell death in several types of cancer cells; however, the precise mechanisms by which MB exerts its anticancer effects remain poorly understood. MB blocked A2780 human ovarian carcinoma cell proliferation both dose- and time-dependently. MB induced apoptosis, with increased poly (ADP-ribose) polymerase (PARP) and caspase-3/9 cleavage. MB also caused autophagy in A2780 cells, with light chain 3 (LC3)-II elevation. Inhibiting MB-induced autophagy with the autophagy inhibitor 3-methyladenine (3-MA) significantly decreased apoptosis, with a reduction of growth inhibition; inhibiting MB-induced apoptosis with the pan-caspase inhibitor Z-VAD-FMK did not decrease autophagy but elevated LC3-II levels, indicating that MB-induced autophagy is cytotoxic and may be upstream of apoptosis. Furthermore, MB increased intracellular reactive oxygen species (ROS) levels, with activated 5′ adenosine monophosphate-activated protein kinase (AMPK), decreased mammalian target of rapamycin (mTOR) and P70S6 kinase phosphorylation, and increased PARP and caspase-3/9 cleavage, and LC3-II elevation; treatment with the ROS scavenger N-acetyl cysteine and the AMPK inhibitor Compound C diminished this effect. Therefore, the ROS/AMPK/mTOR pathway mediates the effect of MB on induction of apoptosis via autophagy in human ovarian carcinoma cells.

Growth enhancing effect of exogenous glycine and characterization of its uptake in halotolerant cyanobacterium Aphanothece halophytica.

Alkaliphilic halotolerant cyanobacterium Aphanothece halophytica showed optimal growth in the medium containing 0.5 M NaCl. The increase of exogenously added glycine to the medium up to 10 mM significantly promoted cell growth under both normal (0.5 M NaCl) and salt stress (2.0 M NaCl) conditions. Salt stress imposed by either 2.0 or 3.0 M NaCl retarded cell growth; however, exogenously added glycine at 10 mM concentration to salt-stress medium resulted in the reduction of growth inhibition particularly under 3.0 M NaCl condition. The uptake of glycine by intact A. halophytica was shown to exhibit saturation kinetics with an apparent K s of 160 μM and V max of 3.9 nmol/min/mg protein. The optimal pH for glycine uptake was at pH 8.0. The uptake activity was decreased in the presence of high concentration of NaCl. Both metabolic inhibitors and ionophores decreased glycine uptake in A. halophytica suggesting an energy-dependent glycine uptake. Several neutral amino acids showed considerable inhibition of glycine uptake with higher than 50 % inhibition observed with serine, cysteine and alanine whereas acidic, basic and aromatic amino acids showed only slight inhibition of glycine uptake.

Abstract B52: The bromodomain inhibitor JQ1 enhances cisplatin-mediated cytotoxicity in ovarian cancer cells

Abstract Ovarian cancer is the most lethal gynecologic malignancy. The treatment of platinum resistance disease remains an urgent and unmet clinical need. Identification of bromodomain inhibitors to disrupt “reading” of aberrant protein acetylation marks in cancer, leading to down-regulation of lineage-specific oncogenes is an exciting development in epigenetic drug therapy. The oncogenic transcription factor c-MYC (MYC) is amplified in approximately 30-40% of high grade serous ovarian tumors and may be epigenetically deregulated via a “super enhancer” on chromosome 8q24. JQ1, an inhibitor of bromodomain binding, down-regulates MYC expression in MYC-amplified tumors. However, this mechanism appears to be tissue-specific and has not been tested as a method for chemosensitization in ovarian cancer. We hypothesized that JQ1 could be used to enhance cisplatin cytotoxicity in ovarian cancer cells, in part by repression of MYC. Ovarian cancer cell lines were selected after in silico evaluation of publically available, annotated cell line collections. Cell lines with relatively low expression of MYC (IGROV-1 and SKOV3) and relatively high expression, A2780 parental [A2780PAR] and isogenic cisplatin-resistant A2780CP20 cells) were treated with 0.1% DMSO, JQ1, cisplatin or the combination of drugs. Sulforhodamine B (SRB) and clonogenic assays were used to assess effects on cell viability. Apoptosis was evaluated by Western Blot detection of cleaved PARP. Expression of MYC was measured by QPCR and Western Blot. JQ1 reduced cell viability and clonogenicity at sub-micromolar concentrations in the ovarian cancer cell lines examined. Furthermore, JQ1 treatment sensitized ovarian cancer cells, including the resistant A2780CP20, to the growth inhibitory and pro-apoptotic effects of cisplatin. However, the cytotoxic effects of JQ1 were not directly correlated with down-regulation of MYC. First, A2780PAR and A2780CP20 cells expressed the highest basal levels of MYC in our panel, but were not the most sensitive to JQ1-induced anti-tumor effects. Second, JQ1 markedly inhibited MYC expression in A2780PAR, but not A2780CP20 cells, despite relatively similar reduction in growth inhibition by JQ1 alone and combined with cisplatin in these cell lines. Taken together, these novel findings suggest that JQ1 could be used to sensitize ovarian cancer cells to platinum-based therapy. Identifying oncogenic targets of JQ1 other than MYC in ovarian cancer is a priority for further development of bromodomain inhibitors in ovarian cancer. Citation Format: Dineo Khabele, Andrew J. Wilson, Jeanette Saskowski, Scott W. Hiebert. The bromodomain inhibitor JQ1 enhances cisplatin-mediated cytotoxicity in ovarian cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr B52.

EVALUATION OF ANTIOXIDANT JUICE CASHEW (Anacardium occidentale Linn) IN Saccharomyces cerevisiae BEFORE AND AFTER DRYING SPRAY

Compounds classified as antioxidants act by inhibiting or reducing the effects caused by free radicals. Several methods have been developed to obtain an evaluation, it is qualitative or quantitative, the antioxidant capacity of various compounds, both by chemical tests or by using biological tests. In this context, this study aimed to evaluate the stability of the antioxidant potential of the juice and the dry material of cashew ((Anacardium ocidentale Linn) before and after spray-drying technique in yeast Saccharomyces cerevisiae proficient and deficient in antioxidant defenses. The juice of A. occidentale L reduced the growth inhibition in all strains of tested, demonstrating its antioxidant potential. Among all four strains of yeast, the variants SOD2Δ WT and SOD showed higher percentages of reduction in growth inhibition before and after drying. Since the mutants SOD1Δ/SOD2Δ and SOD1Δ the substance tested had low antioxidant activity. The juice of pseudofruit of A. occidentale L showed antioxidant potential by increasing the survival of yeast, verified in the reduction of damage caused by hydrogen peroxide, before and after spray drying, which shows that the technique of spray drying did not alter the antioxidant potential of the material analyzed.

A quantitative ELISA for bioactive anti-Nogo-A, a promising regenerative molecule for spinal cord injury repair

The detection and quantification of bioactive anti-Nogo-A mAbs, which is of interest for the treatment of spinal cord injury, has previously been accomplished using cellular or indirect immunoassays. In one such assay the presence of Nogo-A inhibits neurite outgrowth from the PC12 neuronal cell line: pre-treatment with anti-Nogo-A overcomes this inhibition and the concentration of anti-Nogo-A is correlated with the reduction in growth inhibition. In the current work we demonstrate the first anti-Nogo-A sandwich ELISA utilizing a Nogo-A fragment in the role of capture agent and the anti-Nogo-A mAb 11c7 as the soluble analyte. Because the Nogo-A fragment contains the amino acid sequence against which 11c7 was raised, we postulate this combination reproduces the native binding mechanism and results in the detection of bioactive anti-Nogo-A. In support of this hypothesis, we have found good agreement between the inhibitory action of the Nogo-A fragment and myelin proteins used in existing PC12 cell assays. Importantly, unlike the several days required for cellular assays the ELISA is a fast and easy to use method for the detection and quantification of bioactive 11c7 in the range of 500–6000 pg/mL.

Enhancing axon regeneration in peripheral nerves also increases functionally inappropriate reinnervation of targets

The specificity of reinnervation of peripheral targets by regenerating motor axons was studied in mice by using retrograde fluorescent tracers applied to the cut ends of the tibial and common fibular nerves after transection and surgical repair of the sciatic nerve. When the nerve ends were aligned and secured with fibrin glue, more motoneurons labeled after application of tracer to the common fibular nerve were found in regions of the spinal cord that normally contain only tibial motoneurons. The magnitude of such inappropriate reinnervation did not change at different times after repair. Intentional misalignment of the cut nerve stumps at the time of repair resulted in more extensive inappropriate reinnervation of the different peripheral targets. If the proximal stump of the cut nerve was electrically stimulated at the time of repair, if the distal stump was treated with chondroitinase ABC, or if both protocols were applied, the number of motoneurons labeled was increased. This increase was accompanied by more extensive reinnervation of inappropriate targets than found after untreated nerve repair. Although alterations in the caudorostral distributions of labeled motoneurons observed were not as great as observed after purposeful misalignment of the cut nerve ends, the topographic relationship between the spinal locations of motoneuron somata and the peripheral targets of their axons is disrupted. Enhancement of motor axon regeneration by induction of growth-promoting signaling pathways, reduction in growth inhibition in the environment of regenerating axons, or both, is accompanied by an increase in the amount of functionally inappropriate reinnervation of peripheral targets.

Decreased growth inhibition by recombinant gamma interferon is associated with increased transforming growth factor-α production in keratinocytes cultured from psoriatic lesions

Keratinocytes from involved psoriatic plaques (PP), uninvolved, clinically symptomless skin of psoriatic patients (PN) and normal healthy skin (NN) have been cultured in a low calcium serum free system for multiple passages. In this way, the keratinocytes were removed from microenvironmental factors present in the skin. While the basal rate of proliferation of the PP, PN and NN keratinocytes was not different, the PP cells produced more transforming growth factor-alpha (TGF-alpha) than NN cells, and the antiproliferative response of PP cells to gamma interferon (IFN-gamma), a product of activated T lymphocytes, was reduced. We studied IFN-gamma because it can inhibit the proliferation of NN keratinocytes, induce their differentiation and the appearance of two immunoregulatory cell surface molecules, HLA-DR and intercellular adherence molecule-I (ICAM-I), and because in another epithelial cell system, epidermal growth factor (EGF) modulates IFN-gamma activity. The mean antiproliferative effects of IFN-gamma at 50,200, and 500 U/ml for the PP group (n = 10) was less compared to the NN group (n = 11); P less than 0.001, while the PN group (n = 5) had a less dramatic, but statistically significant, reduction in growth inhibition by IFN-gamma only at 200 and 500 U/ml compared to NN cells; P less than 0.05 and P less than 0.01, respectively. The amount of TGF-alpha produced and secreted by PP keratinocytes from five different individuals was significantly greater than by NN keratinocyte cultures. In addition, IFN-gamma induced TGF-alpha to a lesser extent in PP keratinocytes compared to NN keratinocyte cultures. Keratinocytes isolated from atopic dermatitis and Sézary syndrome patients were similar to NN keratinocytes. In contrast to its differential effects on TGF-alpha production and proliferation, IFN-gamma induced similar amounts of HLA-DR and ICAM-I on PP, PN and NN keratinocytes. Thus, for the PP keratinocytes, there was a dissociation between the antiproliferative and immunomodulatory effects of IFN-gamma. These results support our previous hypothesis that the hyperproliferation and altered differentiation of keratinocytes in psoriatic plaques is linked to an altered responsiveness of the keratinocytes to IFN-gamma. Moreover, these results provide an in vitro correlate of our in vivo observation of increased TGF-alpha levels in psoriatic plaques. A new pathophysiological model to understand psoriasis is proposed which integrates these observations involving IFN-gamma and TGF-alpha. This experimental approach also provides a system to dissect biochemical pathways of pathophysiological importance for keratinocyte hyperproliferation in psoriasis.

Differential reduction by caffeine of adriamycin induced cell killing and cell cycle delays in chinese hamster v79 cells

Exponentially growing Chinese-hamster V79-cells were treated with various doses of adriamycin (ADR) for 1 hr in the presence or absence of 2 mM caffeine and were subsequently incubated for 24 hr in fresh medium with or without caffeine (2 mM) before plating to assay for survival. The results indicated a reduction in killing when caffeine was present during treatment with ADR (e.g., reduction in killing from 0.03 to 0.3 after exposure to 0.5 μ/ml ADR). This reduction in killing was even more pronounced after a 24 hr treatment with ADR in the presence of caffeine (e.g, reduction from 0.005 to OS after exposure to 0.08 μg/ml ADR). Incubation with caffeine after ADR treatment (1 hr) caused only a comparably small increase in cell survival. Presence of caffeine either simultaneously or after treatment with ADR caused a reduction of the inhibition of growth and mean-cell-volume increase, and a reduction of the accumulation of cells in G2-phase. Qualitatively similar results were also obtained after continuous treatment with ADR in the presence or absence of caffeine. Reduction in growth inhibition and accumulation of cells in G2-phase was observed under conditions only slightly affecting cell survival, thus suggesting that caffeine may affect these two phenomena by independent mechanisms. Flow cytometry measurement of the intracellular ADR content indicated a reduction in the presence of caffeine. Furthermore, post-treatment incubation with caffeine was found to increase the rate of decay of ADR-related fluorescence. Quantitative comparison between the effect of caffeine in the intracellular ADR accumulation and cell survival suggested that the observed reduction in killing could be attributed to a decrease in the intracellular drug levels. The reduction by caffeine of the ADR-induced cell cycle delays is attributed to either the decrease in the intracellular ADR dose in the presence of caffeine, or to an effect of caffeine similar to that exerted after exposure of cells to ionizing radiation. Trifluoperazine, which had only a small effect on cell survival of cells treated with ADR alone, potentiated killing when cells were treated with ADR in the presence of caffeine. This effect can be partly attributed to the observed modification in the intracellular ADR content under these conditions but, as a quantitative comparison suggests, other effects might also be involved.

Biochemical and Antibacterial Properties of Bovine Mammary Secretion During Mammary Involution and at Parturition

Mammary secretions from four Holstein Friesian cows were collected during late lactation, early involution, and early in subsequent lactation. Changes of pH, concentration of serum albumin, immunoglobulin G, citrate, lactoferrin, and number of leukocytes in secretions were typical of milk from glands undergoing these physiological transitions. Whey prepared from a cow's secretions was evaluated for its capacity to inhibit growth of Escherichia coli, 60-Lilly, and a coliform strain isolated from mammary secretions of that cow. Wheys from different glands of the same cow differed markedly in their capacity to inhibit growth of coliforms. Inhibition of both strains by the wheys increased significantly during the dry period and was maximal in wheys collected day 15 of the dry period and at parturition in the subsequent lactation. The coliform strain isolated from a specific cow was inhibited more than Escherichia coli, 60-Lilly, by whey from the specific cow. Inhibition of the cow-specific coliform strain by the day 15 whey was reduced significantly after addition of ferric iron or sodium citrate. Addition of excess ferric iron or citrate to wheys collected at parturition did not alter significantly their inhibitory capacity within cow. However, reduction of growth inhibition was significant when data from the four cows were pooled.