Interaction of Gold Nanoparticles with Rat Brain Synaptosomal Plasma Membrane Na+/K+ - Atpase and Mg2+-Atpase

The aim of the work was to investigate the interaction between borate capped gold nanoparticles (NPs) and the rat brain synaptosomal plasma membranes (SPM), as well as the effects of these NPs on SPM Na+/K+ - ATPase and Mg2+-ATPase activity. The changes in the UV-vis spectra of NPs and SPM assembly suggested the agglomeration and precipitation of NPs. FTIR measurements indicated that both protein -SH and -NH2 groups and positively charged membrane fragments were implicated in the adhesion of SPM to the surface of NPs. AFM showed an increase in the particularization of the SPM material after mixing with gold NPs. Influence of gold NPs on Na+/K+-ATPase and Mg2+-ATPase activity was investigated as the function of NPs and protein concentration, preincubation time and also in the presence of various concentrations of ouabain, the specific enzyme inhibitor. NPs induced the stimulation of Na+/K+-ATPase activity for more than 100%, since Mg2+-ATPase activity reminded unaffected. We propose that this stimulation of enzyme activity was a consequence of an increase of the active surface of membranes

Interaction of Gold Nanoparticles with Rat Brain Synaptosomal Plasma Membrane Na+/K+ - Atpase and Mg2+-Atpase

The aim of the work was to investigate the interaction between borate capped gold nanoparticles (NPs) and the rat brain synaptosomal plasma membranes (SPM), as well as the effects of these NPs on SPM Na+/K+ - ATPase and Mg2+-ATPase activity. The changes in the UV-vis spectra of NPs and SPM assembly suggested the agglomeration and precipitation of NPs. FTIR measurements indicated that both protein -SH and -NH2 groups and positively charged membrane fragments were implicated in the adhesion of SPM to the surface of NPs. AFM showed an increase in the particularization of the SPM material after mixing with gold NPs. Influence of gold NPs on Na+/K+-ATPase and Mg2+-ATPase activity was investigated as the function of NPs and protein concentration, preincubation time and also in the presence of various concentrations of ouabain, the specific enzyme inhibitor. NPs induced the stimulation of Na+/K+-ATPase activity for more than 100%, since Mg2+-ATPase activity reminded unaffected. We propose that this stimulation of enzyme activity was a consequence of an increase of the active surface of membranes

Changes in cellular signaling proteins in extracts from A549, H460, and U2OS cells treated with cisplatin or docetaxel

Cell extracts from A549, H460, and U2OS human cancer cell lines treated with cisplatin and docetaxel were analyzed by mass spectrometry (MS) proteomic analysis. The extracts were enriched for cellular signaling proteins using a mix of three different immobilized kinase inhibitors (Purvalanol B, Bisindolylmaleimide X, and (R)-3-(4-((1-Phenylethyl)amino)thieno[2,3-d]pyrimidin-6-yl)benzoic acid (SB6-060-05)) on sepharose bead columns. Raw data is deposited in the PRIDE database [1], project number PXD005286. Data presented (Table 1) shows changes relative to untreated control for each biological replicate for the three cell lines

Targeting the non-canonical roles of PCNA modifies and increases the response to targeted anti-cancer therapy

Receptor tyrosine kinases (RTKs), such as HER2 and/or EGFR are important therapeutic targets in multiple cancer cells. Low and/or short response to targeted therapies are often due to activation of compensatory signaling pathways, and therefore a combination of kinase inhibitors with other anti-cancer therapies have been proposed as promising strategies. PCNA is recently shown to have non-canonical cytosolic roles, and targeting PCNA with a cell-penetrating peptide containing the PCNA-interacting motif APIM is shown to mediate changes in central signaling pathways such as PI3K/Akt and MAPK, acting downstream of multiple RTKs. In this study, we show how targeting PCNA increased the anti-cancer activity of EGFR/HER2/VEGFR inhibition in vitro as well as in vivo. The combination treatment resulted in reduced tumor load and increased the survival compared to either single agent treatments. The combination treatment affected multiple cellular signaling responses not seen by EGFR/HER2/VEGFR inhibition alone, and changes were seen in pathways determining protein degradation, ER-stress, apoptosis and autophagy. Our results suggest that targeting the non-canonical roles of PCNA in cellular signaling have the potential to improve targeted therapies

Changes in cellular signaling proteins in extracts from A549, H460, and U2OS cells treated with cisplatin or docetaxel

Cell extracts from A549, H460, and U2OS human cancer cell lines treated with cisplatin and docetaxel were analyzed by mass spectrometry (MS) proteomic analysis. The extracts were enriched for cellular signaling proteins using a mix of three different immobilized kinase inhibitors (Purvalanol B, Bisindolylmaleimide X, and (R)-3-(4-((1-Phenylethyl)amino)thieno[2,3-d]pyrimidin-6-yl)benzoic acid (SB6-060-05)) on sepharose bead columns. Raw data is deposited in the PRIDE database [1], project number PXD005286. Data presented (Table 1) shows changes relative to untreated control for each biological replicate for the three cell lines

In vitro effects of some gold complexes on Na+/K+ ATPase activity and cell proliferation

The in vitro influence of gold(III) complexes, H[AuCl4], [Au(DMSO)(2)Cl-2]Cl and [Au(bipy)Cl-2]Cl (bipy = 2,2-bipyridine), upon commercially available Na+ /K+ ATPase activity, purified from porcine brain cortex, was investigated. Additionally, the complexes were tested on human lymphocytes, and incidence of micronuclei and cell proliferation index was determined. Concentration-dependent inhibition of the enzyme for all three compounds was obtained, but with differing potencies. Calculated IC50 from Hill analysis were (in M): 5.75 x 10(-7), 5.50 x 10(-6) and 3.98 x 10(-5), for H[AuCl4], [Au(DMSO)(2)Cl-2]Cl and [Au(bipy)Cl-2]Cl, respectively, while Hill coefficient values, n, were above 1 in all cases. This inhibition can be prevented using -SH donating ligands such as L-Cys and glutathione, and these ligands can also cause a recovery of the enzyme activity after the induced inhibition. Kinetic analysis demonstrated that each of the studied gold(III) complexes affects Na+ /K+ ATPase reducing maximum enzymatic velocity, V-max, but not significantly changing the affinity for the substrate (K-M value), implying a noncompetitive mode of the interaction. Furthermore, among investigated gold(III) complexes, the [Au(bipy)Cl-2]Cl complex exhibits a strong cytotoxic effect on human lymphocytes, which suggests its potential for use in antitumor therapy. (C(C) 2013 Elsevier Inc. All rights reserved

Inhibition of Na+/K+-ATPase and cytotoxicity of a few selected gold(III) complexes

Na+/K+-ATPase is in charge of maintaining the ionic and osmotic intracellular balance by using ATP as an energy source to drive excess Na+ ions out of the cell in exchange for K+ ions. We explored whether three representative cytotoxic gold(III) compounds might interfere with Na+/K+-ATPase and cause its inhibition at pharmacologically relevant concentrations. The tested complexes were [Au(bipy)(OH)(2)][PF6] (bipy = 2,2-bipyridine), [Au (py(dmb)-H)(CH3COO)(2)] (py(dmb)-H = deprotonated 6-(1,1-dimethylbenzyl)-pyridine), and [Au(bipy(dmb)-H)(OH)][PF6] (bipy(dmb)-H = deprotonated 6-(1,1-dimethylbenzyl)-2,2-bipyridine). We found that all of them caused a pronounced and similar inhibition of Na+/K+-ATPase activity. Inhibition was found to be non-competitive and reversible. Remarkably, treatment with cysteine resulted in reversal or prevention of Na+/K+-ATPase inhibition. It is very likely that the described effects may contribute to the overall cytotoxic profile of these gold complexes. (C) 2014 Elsevier Inc. All rights reserved

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Anti-Cancer Potential of Homemade Fresh Garlic Extract Is Related to Increased Endoplasmic Reticulum Stress

The use of garlic and garlic-based extracts has been linked to decreased incidence of cancer in epidemiological studies. Here we examine the molecular and cellular activities of a simple homemade ethanol-based garlic extract (GE). We show that GE inhibits growth of several different cancer cells in vitro, as well as cancer growth in vivo in a syngeneic orthotopic breast cancer model. Multiple myeloma cells were found to be especially sensitive to GE. The GE was fractionated using solid-phase extractions, and we identified allicin in one GE fraction; however, growth inhibitory activities were found in several additional fractions. These activities were lost during freeze or vacuum drying, suggesting that the main anti-cancer compounds in GE are volatile. The anti-cancer activity was stable for more than six months in −20 °C. We found that GE enhanced the activities of chemotherapeutics, as well as MAPK and PI3K inhibitors. Furthermore, GE affected hundreds of proteins involved in cellular signalling, including changes in vital cell signalling cascades regulating proliferation, apoptosis, and the cellular redox balance. Our data indicate that the reduced proliferation of the cancer cells treated by GE is at least partly mediated by increased endoplasmic reticulum (ER) stress

"Two hits - one stone" increased efficacy of cisplatin-based therapies by targeting PCNA's role in both DNA repair and cellular signaling

Low response rate and rapid development of resistance against commonly used chemotherapeutic regimes demand new multi-targeting anti-cancer strategies. In this study, we target the stress-related roles of the scaffold protein PCNA with a cell-penetrating peptide containing the PCNA-interacting motif APIM. The APIM-peptide increased the efficacy of cisplatin-based therapies in a muscle-invasive bladder cancer (MIBC) solid tumor model in rat and in bladder cancer (BC) cell lines. By combining multiple omics-levels, from gene expression to proteome/kinome and metabolome, we revealed a unique downregulation of the EGFR/ERBB2 and PI3K/Akt/mTOR pathways in the APIM-peptide-cisplatin combination treated cells. Additionally, the combination treatment reduced the expression of anti-apoptotic proteins and proteins involved in development of resistance to cisplatin. Concurrently, we observed increased levels of DNA breaks in combination treated cells, suggesting that the APIM-peptide impaired PCNA - DNA repair protein interactions and reduced the efficacy of repair. This was also seen in cisplatin-resistant cells, which notably was re-sensitized to cisplatin by the APIM-peptide. Our data indicate that the increased efficacy of cisplatin treatment is mediated both via downregulation of known oncogenic signaling pathways and inhibition of DNA repair/translesion synthesis (TLS), thus the APIM-peptide hits both nuclear and cytosolic functions of PCNA. The novel multi-targeting strategy of the APIM-peptide could potentially improve the efficacy of chemotherapeutic regiments for treatment of MIBC, and likely other solid tumors.publishedVersionCopyright: Søgaard et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited