The role of the calmodulin-binding and calmodulin-like domains of the epidermal growth factor receptor in tyrosine kinase activation.

The epidermal growth factor receptor (EGFR) harbors a calmodulin (CaM)-binding domain (CaM-BD) and a CaM-like domain (CaM-LD) upstream and downstream, respectively, of the tyrosine kinase (TK) domain. We demonstrate in this paper that deletion of the positively charged CaM-BD (EGFR/CaM-BD∆) inactivated the TK activity of the receptor. Moreover, deletion of the negatively charged CaM-LD (EGFR/CaM-LD∆), leaving a single negative residue (glutamate), reduced the activity of the receptor. In contrast, substituting the CaM-LD with a histidine/valine-rich peptide (EGFR/InvCaM-LD) caused full inactivation. We also demonstrated using confocal microscopy and flow cytometry that the chimera EGFR-green fluorescent protein (GFP)/CaM-BD∆, the EGFR/CaM-LD∆, and EGFR/InvCaM-LD mutants all bind tetramethylrhodamine-labelled EGF. These EGFR mutants were localized at the plasma membrane as the wild-type receptor does. However, only the EGFR/CaM-LD∆ and EGFR/InvCaM-LD mutants appear to undergo ligand-dependent internalization, while the EGFR-GFP/CaM-BD∆ mutant seems to be deficient in this regard. The obtained results and in silico modelling studies of the asymmetric structure of the EGFR kinase dimer support a role of a CaM-BD/CaM-LD electrostatic interaction in the allosteric activation of the EGFR TK.

Simultaneous cleanup of Reactive Black 5 and cadmium by a desert soil bacterium.

Multi-contaminated industrial wastewaters pose serious environmental risks due to high toxicity and non-biodegradability. The work reported here evaluated the ability of Pseudomonas aeruginosa strain Gb30 isolated from desert soil to simultaneously remove cadmium (Cd) and Reactive Black 5 (RB5), both common contaminants in various industrial effluents. The strain was able to grow normally and decolorize 50 mg L-1 RB5 within 24 h of incubation in the presence of 0.629 m mol L-1 of Cd2+. In order to evaluate strain performance in RB5 detoxification, a cytotoxicity test using Human Embryonic Kidney cells (HEK293) was used. Cadmium removal from culture media was determined using atomic adsorption. Even in presence of (0.115 + 0.157 + 0.401 + 0.381) m mol L-1, respectively, of Cr6+, Cd2+, Cu2+ and Zn2+ in the growth medium, strain Gb30 successfully removed 35% of RB5 and 44%, 36%, 59% and 97%, respectively, of introduced Zn2+, Cu2+, Cr6+ and Cd2+, simultaneously. In order to understand the mechanism of Cd removal used by P. aeruginosa strain Gb30, biosorption and bioaccumulation abilities were examined. The strain was preferentially biosorbing Cd on the cell surface, as opposed to intracellular bioaccumulation. Microscopic investigations using AFM, SEM and FTIR analysis of the bacterial biomass confirmed the presence of various structural features, which enabled the strain to interact with metal ions. The study suggests that Pseudomonas aeruginosa Gb30 is a potential candidate for bioremediation of textile effluents in the presence of complex dye-metal contamination.

Chemical composition, anti-biofilm activity and potential cytotoxic effect on cancer cells of Rosmarinus officinalis L. essential oil from Tunisia.

BACKGROUND: Rosmarinus officinalis L. from Tunisia, popularly known as rosemary, is of a considerable importance for its medicinal uses and aromatic value. The aim of this study was to examine the chemical composition of Rosmarinus officinalis essential oil (ROEO) and to evaluate its antibiofilm activity on biofilm-forming bacterium and its anticancer activity on cancer cell lines. METHODS: The chemical composition of Rosmarinus officinalis essential oil (ROEO) was analyzed by GC-MS and its antibacterial activity was evaluated by micro-dilution method. The antibofilm activity of ROEO was evaluated using the crystal violet test and the cytotoxicity activity was determined by the MTT assay. RESULTS: In this research, thirty-six compounds were identified in ROEO using GC-MS analyses. The main components were 1,8-cineole (23.56%), camphene (12.78%), camphor (12.55%) and ß-pinene (12.3%). The antibacterial activity of ROEO was evaluated by micro-dilution method. The oil exhibited inhibition and bactericidal effect against two strains: Staphylococcus aureus ATCC 9144 and Staphylococcus epidermidis S61. It was found that the minimum inhibitory concentration (MIC) obtained for S. aureus and S. epidermidis ranged from 1.25 to 2.5 and from 0.312 to 0.625 µl ml-1, respectively and the minimum bactericidal concentration (MBC) were in the order of 5 and 2.5 µl ml-1, respectively. Furthermore, this oil showed a S. epidermidis biofilm inhibition more than 57% at a concentration of 25 µl ml-1. The eradication of 67% of the established biofilm was observed at a concentration of 50 µl ml-1 of ROEO, whereas the dose of 25 µl ml-1 removed only 38% of preformed biofilm. ROEO strongly inhibited the proliferation of Hela and MCF-7 cells with IC50 values of 0.011 and 0.253 µl ml-1, respectively. CONCLUSION: Our results demonstrate that ROEO could have a potential role in the treatment of diseases related to infection by microorganisms or proliferation of cancer cells.

Controversial effect on Erk activation of some cytotoxic drugs in human LOVO colon cancer cells.

UNLABELLED: The use of some classic antibiotics was recently shown to inhibit growth and to induce apoptosis in human LOVO colon cancer cells. In this study, we describe that ciprofloxacin (CI), trimebutine maleate (COL) and tiemonium methylsulfate (VIS) greatly inhibit cell proliferation in vitro. Proliferation inhibition reached its maximum at 10(-4 )M, 10(-3 )M and 10(-2 )M, respectively, for COL, CI and VIS. Moreover, phospho-extracellular-regulated kinase was totally abrogated in non-apoptotic cytotoxicity of VIS but decreases or increases in the apoptotic inhibition, respectively, of COL and CI treatments. ABBREVIATIONS: CI: ciprofloxacin; COL: trimebutine maleate; VIS: tiemonium methylsulfate; MAPK/Erk: mitogen-activated protein kinases/extracellular-regulated kinase.

Effect of estradiol and clomiphene citrate on Erk activation in breast cancer cells.

The mitogen-activated protein kinase (MAPK) signaling pathway plays key roles in the transmission of proliferative signals in normal and dysregulated cells. Nevertheless, some studies have shown that activation of the extracellular regulated kinases 1/2 (Erk1/2) is involved in apoptosis. In this study, we evaluate the effect of two fertilizing drugs, clomiphene citrate and estradiol, on the activation of Erk1/2 and the viability of two breast cancer cell lines, MCF-7 (hormone dependent) and BT20 (hormone independent).We show that both drugs induce Erk1/2 phosphorylation in MCF-7 and BT20 cells despite their opposite effect on cell viability. In fact, clomiphene citrate is significantly proapoptotic while estradiol promotes cell proliferation. The fact that phospho-Erk1/2 is a common element to both mechanisms suggests that specific factors deciding between proliferation and apoptosis must be operative downstream of this signaling pathway.

A premature termination of human epidermal growth factor receptor transcription in Escherichia coli.

Our success in producing an active epidermal growth factor receptor (EGFR) tyrosine kinase in Escherichia coli encouraged us to express the full-length receptor in the same host. Despite its large size, we were successful at producing the full-length EGFR protein fused to glutathione S-transferase (GST) that was detected by Western blot analysis. Moreover, we obtained a majoritarian truncated GST-EGFR form detectable by gel electrophoresis and Western blot. This truncated protein was purified and confirmed by MALDI-TOF/TOF analysis to belong to the N-terminal extracellular region of the EGFR fused to GST. Northern blot analysis showed two transcripts suggesting the occurrence of a transcriptional arrest.

In vitro activation and inhibition of recombinant EGFR tyrosine kinase expressed in Escherichia coli.

The present work concerns the heterologous expression of the intracellular domain harbouring the tyrosine kinase activity of the epidermal growth factor receptor (EGFR). Protein expression was improved thanks to the deletion of a 13-amino acid peptide of the juxtamembrane region (JM). The recombinant proteins were produced as a glutathione S-transferase (GST) fusion in Escherichia coli, and the solubilisation was performed by sarkosyl addition during extraction. The produced proteins spontaneously dimerize allowing the activation of the tyrosine kinase domain in the presence of [γ-(32)P]ATP. The activity assay has revealed the autophosphorylation of EGFR proteins which was decreased in the presence of genistein. Our system could facilitate the screening of EGFR inhibitors without the need of adding an exogenous substrate.

Toxicity evaluation of synthetic glucocorticoids against breast cancer cell lines MDA-MB-231, MCF-7 and human embryonic kidney HEK293.

In this study, we conducted a comprehensive assessment of the cytotoxicity of three glucocorticoids, namely Hydrocortisone, Dexamethasone, and Methylprednisolone, using three different human cell lines: MDA-MB-231, MCF-7 (both adenocarcinoma cell lines), and HEK293 (kidney epithelial cell line). At lower concentrations exceeding 50 µM, we did not observe any significant toxic effects of these glucocorticoids. However, when exposed to higher concentrations, Hydrocortisone exhibited dose-dependent cytotoxic effects on all three cell lines, with calculated IC50 values of 12 ± 0.6 mM for HEK293, 2.11 ± 0.05 mM for MDA-MB-231, and 2.73 ± 0.128 mM for MCF-7 cells after 48 h of exposure. Notably, Hydrocortisone, at its respective IC50 concentrations, demonstrated an inhibitory effect on the proliferation of the cancer cell lines, as evidenced by a substantial reduction in BrdU absorbance in a dose-dependent manner, coupled with a markedly reduced rate of colony formation in treated cells. Furthermore, Hydrocortisone exhibited remarkable anti-migratory properties in MDA-MB-231 and MCF-7 cells, and it induced cell cycle arrest in the SubG1 phase in MDA-MB-231 cells. In addition to these effects, Hydrocortisone triggered apoptosis in both cancer cell types, leading to observable morphological changes. This apoptotic response was characterized by a significant increase in the activity of caspase-3, which was time-dependent. Additionally, Hydrocortisone downregulated the expression of anti-apoptotic Bcl-2 proteins. In summary, our findings underscore the safety of clinical doses in terms of cell toxicity meanwhile increased concentration were showing an anti-proliferative potential of Hydrocortisone, particularly against adenocarcinoma breast cancer cell lines.

Chemical alternative for cell identification and cross-contamination detection.

Misidentification of human cell lines has previously led to confusing results during cell culture experiments. Although several enzymatic as well as molecular analysis approaches have been developed for cell-line authentication, these methods remain costly. In the present paper, we describe a simple chemical alternative based on known compound cell cytotoxicity. In addition to cisplatin, a pool of eight tamoxifen derivative compounds was used to compare the cytotoxic effects on three different breast cancer cell lines: MCF-7, T47D and MDA-MB-231. Our results show that four out of the eight cytotoxic-related compounds allowed to distinguish the different cell lines based on their IC50 (the half maximal inhibitory concentration) values which are cell type dependent. The remaining chemicals, particularly the most cytotoxic P15, showed close IC50 values for all the cell lines. Interestingly, flow cytometry experiments have identified notable differences among the three cell lines treated with P15. T47D and MDA-MB231 cells were blocked in SubG1 phase and S phase, respectively, while no significant change in cell cycle profile was noticed for MCF-7 cells. Differences were also noted at the level of caspase-3 activity and cell proliferation in P15-treated cells.

The conservative effects of lipopeptides from Bacillus methylotrophicus DCS1 on sunflower oil-in-water emulsion and raw beef patties quality.

Lipid oxidation was considered as a problem in food conservation. The present study aims to investigate the effect of lipopeptides DCS1 on the conservation of food models against lipid oxidation by determining the primary and the secondary oxidation products. Lipopeptides DCS1 are able to preserve the nutritional properties of the emulsion during 23 days of storage, at a concentration of 0.0125% (w/w of emulsion), by slowing down the formation of hydroperoxides and malondialdehyde (MDA) compounds. The direct incorporation of lipopeptides in ground beef patties at a concentration of 0.5% (w/w of meat) was found to be more effective than gelatin film enriched with lipopeptides (2.5%, w/w of gelatin) as a coating, in inhibiting lipid oxidation. Furthermore, lipopeptides DCS1 are not toxic to human kidney cells HEK293 up to a concentration of 250 µg/ml. The results indicate that lipopeptides DCS1 are effective for the preservation of fatty foods against lipid oxidation.

Anti-Diarrheal Drug Repositioning in Tumour Cell Cytotoxicity.

BACKGROUND: Drug repositioning is becoming an ideal strategy to select new anticancer drugs. In particular, drugs treating the side effects of chemotherapy are the best candidates. OBJECTIVE: In this present work, we undertook the evaluation of anti-tumour activity of two anti-diarrheal drugs (nifuroxazide and rifaximin). METHODS: Anti-proliferative effect against breast cancer cells (MDA-MB-231, MCF-7 and T47D) was assessed by MTT analysis, the Brdu incorporation, mitochondrial permeability and caspase-3 activity. RESULTS: Both the drugs displayed cytotoxic effects on MCF-7, T47D and MDA-MB-231 cells. The lowest IC50 values were obtained on MCF-7 cells after 24, 48 and 72 hours of treatment while T47D and MDA-MB-231 were more resistant. The IC50 values on T47D and MDA-MB-231 cells became significantly low after 72 hours of treatment showing a late cytotoxicity effect especially of nifuroxazide but still less important than that of MCF-7 cells. According to the IC50 values, the non-tumour cell line HEK293 seems to be less sensitive to cytotoxicity especially against rifaximin. Both the drugs have shown an accumulation of rhodamine 123 as a function of the rise of their concentrations while the Brdu incorporation decreased. Despite the absence of a significant difference in the cell cycle between the treated and non-treated MCF-7 cells, the caspase-3 activity increased with the drug concentrations rise suggesting an apoptotic effect. CONCLUSION: Nifuroxazide and rifaximin are used to overcome the diarrheal side effect of anticancer drugs. However, they have shown to be anti-tumour drugs which make them potential dual effective drugs against cancer and the side effects of chemotherapy.