RANKL, OPG, and RUNX2 expression and epigenetic modifications in giant cell tumour of bone in 32 patients.

Aims: The present study investigated receptor activator of nuclear factor kappa-Β ligand (RANKL), osteoprotegerin (OPG), and Runt-related transcription factor 2 (RUNX2) gene expressions in giant cell tumour of bone (GCTB) patients in relationship with tumour recurrence. We also aimed to investigate the influence of CpG methylation on the transcriptional levels of RANKL and OPG. Methods: A total of 32 GCTB tissue samples were analyzed, and the expression of RANKL, OPG, and RUNX2 was evaluated by quantitative polymerase chain reaction (qPCR). The methylation status of RANKL and OPG was also evaluated by quantitative methylation-specific polymerase chain reaction (qMSP). Results: We found that RANKL and RUNX2 gene expression was upregulated more in recurrent than in non-recurrent GCTB tissues, while OPG gene expression was downregulated more in recurrent than in non-recurrent GCTB tissues. Additionally, we proved that changes in DNA methylation contribute to upregulating the expression of RANKL and downregulating the expression of OPG, which are critical for bone homeostasis and GCTB development. Conclusion: Our results suggest that the overexpression of RANKL/RUNX2 and the lower expression of OPG are associated with recurrence in GCTB patients.

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.

Three Component One-Pot Synthesis and Antiproliferative Activity of New [1,2,4]Triazolo[4,3-a]pyrimidines.

A series of new [1,2,4]triazolo[4,3-a]pyrimidine derivatives was prepared using a one-pot three-component synthesis from 5-amino-1-phenyl-1H-1,2,4-triazoles, aromatic aldehydes and ethyl acetoacetate. The compound structures were confirmed by IR, 1H-NMR, 13C-NMR, HRMS and X-ray analyses. The biological activity of these compounds as antitumor agents was evaluated. Their antitumor activities against cancer cell lines (MDA-MB-231 and MCF-7) were tested by the MTT in vitro method. Among them, compounds 4c and 4j displayed the best antitumor activity with IC50 values of 17.83 µM and 19.73 µM against MDA-MB-231 and MCF-7 cell lines, respectively, compared to the Cisplatin reference.

Benzochromenopyrimidines: Synthesis, Antiproliferative Activity against Colorectal Cancer and Physicochemical Properties.

Ten new differently substituted 3-benzyl-5-aryl-3,5-dihydro-4H-benzo[6,7]chromeno[2,3-d]pyrimidin-4,6,11-triones 3 were synthesized by a simple and cost-efficient procedure in a one-pot, three-component reaction from readily available ethyl 2-amino-4-aryl-5,10-dioxo-5,10-dihydro-4H-benzo[g]chromene-3-carboxylates, benzylamine and triethyl orthoformate under solvent- and catalyst-free conditions. All the new compounds were screened for their antiproliferative activity against two colorectal-cancer-cell lines. The results showed that the compounds 3-benzyl-5-phenyl-3,5-dihydro-4H-benzo[6,7]chromeno[2,3-d]pyrimidine-4,6,11-trione (3a) and 3-benzyl-5-(3-hydroxyphenyl)-3,5-dihydro-4H-benzo[6,7]chromeno[2,3-d]pyrimidine-4,6,11-trione (3g) exhibited the most potent balanced inhibitory activity against human LoVo and HCT-116 cancer cells.

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.

Antibacterial, antibiofilm and cytotoxic properties of prodigiosin produced by a newly isolated Serratia sp. C6LB from a milk collection center.

The aim of this work was to produce a red tripyrrole pigment prodigiosin from Serratia sp. C6LB strain, to investigate the promising antimicrobial properties on Gram-positive and Gram-negative bacterial strains. The research was also proposed to evaluate the antibiofilm activity on Staphylococcus epidermidis S61 biofilm and its cytotoxic activity against human cancer cell lines. The production and structural elucidation of prodigiosin was carried out using spectrophotometric scanning, TLC, HPLC, FTIR and NMR analysis. The pigment production was optimized using mannose and peptone as carbon and nitrogen sources, respectively. The study confirmed promising antibacterial properties of prodigiosin on eight Gram-positive and Gram-negative bacterial strains with MICs values ranged from 0.039 to 2.5 mg/mL. Antiadhesive activity test of prodigiosin on Staphylococcus epidermidis S61 biofilm exhibited 99.9% inhibition, whereas maximum biofilm eradication activity reached 65%. Cytotoxic activity showed IC50 of 16 µg/mL and 6.7 µg/mL against breast cancer lines MCF-7 and MDA-MB231, respectively.

Molecular insights into MYO3A kinase domain variants explain variability in both severity and progression of DFNB30 hearing impairment.

Hereditary hearing impairment (HI) is a common disease with the highest incidence among sensory defects. Several genes have been identified to affect stereocilia structure causing HI, including the unconventional myosin3A. Interestingly, we noticed that variants in MYO3A gene have been previously found to cause variable HI onset and severity. Using clinical exome sequencing, we identified a novel pathogenic variant p.(Lys50Arg) in the MYO3A kinase domain (MYO3A-KD). Previous in vitro studies supported its damaging effect as a 'kinase-dead' mutant. We further analyzed this variation through molecular dynamics which predicts that changes in flexibility of MYO3A structure would influence the protein-ATP binding properties. This Lys50Arg mutation segregated with congenital profound non-syndromic HI. To better investigate this variability, we collected previously identified MYO3A-KDs variants, p.(Tyr129Cys), p.(His142Gln) and p.(Pro189Thr), and built both wild type and mutant 3 D MYO3A-KD models to assess their impact on the protein structure and function. Our results suggest that KD mutations could either cause a congenital profound form of HI, when particularly affecting the kinase activity and preventing the auto-phosphorylation of the motor, or a late onset and progressive form, when partially or completely inactivating the MYO3A protein. In conclusion, we report a novel pathogenic variant affecting the ATP-binding site within the MYO3A-KD causing congenital profound HI. Through computational approaches we provide a deeper understanding on the correlation between the effects of MYO3A-KD mutations and the variable hearing phenotypes. To the best of our knowledge this is the first study to correlate mutations' genotypes with the variable phenotypes of DFNB30.Communicated by Ramaswamy H. Sarma.

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.

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.

Antibacterial, anti-adherent and cytotoxic activities of surfactin(s) from a lipolytic strain Bacillus safensis F4.

The bacterial strain F4, isolated from olive oil-contaminated soil, has been found to produce biosurfactants as confirmed by oil displacement test and the emulsification index results. The identification of the strain F4, by 16S ribosomal RNA gene, showed a close similarity to Bacillus safensis, therefore the strain has been termed Bacillus safensis F4. The Thin Layer Chromatography (TLC) and the High Pressure Liquid Chromatography-Mass Spectrometry (HPLC-MS/MS) demonstrated that the biosurfactant had a lipopeptide structure and was classified as surfactin. The present study showed also that the produced biosurfactant has an important antibacterial activity against several pathogen strains as monitored with minimum inhibitory concentration (MIC) micro-assays. In particular, it presented an interesting anti-planktonic activity with a MIC of 6.25 mg mL-1 and anti-adhesive activity which exceeded 80% against the biofilm-forming Staphylococcus epidermidis S61 strain. Moreover, the produced lipopeptide showed an antitumor activity against T47D breast cancer cells and B16F10 mouse melanoma cells with IC50 of 0.66 mg mL-1 and 1.17 mg mL-1, respectively. Thus, our results demonstrated that Bacillus safensis F4 biosurfactant exhibited a polyvalent activity via a considerable antibiofilm and antitumoral potencies.

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.

Cumin (Cuminum cyminum L.) from traditional uses to potential biomedical applications.

Cumin (Cuminum cyminum L.) is a small annual and herbaceous plant belonging to the Apiaceae family. It is a multipurpose plant species cultivated in the Middle East, India, China, and several Mediterranean countries, including Tunisia. Its fruit, known as cumin seed, is most widely used for culinary and medicinal purposes. It is generally used as a food additive, popular spice, and flavoring agent in many cuisines. Cumin has also been widely used in traditional medicine to treat a variety of diseases, including hypolipidemia, cancer, and diabetes. The literature presents ample evidence for the biological and biomedical activities of cumin, which have generally been ascribed to its content and action of its active constituents, such as terpens, phenols, and flavonoids. The present paper provides an overview of phytochemical profile, biological activities, and ethnomedical and pharmacological uses of Cumin.

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.

Selection of a suitable disc bioassay for the screening of anti-tumor molecules.

The crown gall induced in potato discs by Agrobacterium tumefaciens is becoming largely utilised in screening anti-tumor agents. The present work is showing that beet discs are more adequate for the anti-tumor screening test. In fact, maximal tumor induction was observed on beet discs (87.5%), followed by carrot discs (75%) and potato discs (68.5%). Beet discs present the most sensibility to crown gall disease with a fast expression of symptoms and more visible galls without any staining need. The beet discs bioassay was carried out by using some synthesized organometallics known for their antitumor activity in mammalian cells. We found significant crown gall inhibition (20.7% to 40.55%) of the tested compounds. Overall results supported that beet bioassay might be a potential prescreen system of anti-tumor molecules in mammalian cells.

Monoclonal antibodies as cancer therapeutics.

Three main targets were subjected for the most approved monoclonal antibodies (mAbs) in cancer therapy: EGFR in solid cancer, the clusters of differentiation in blood cancer and VEGF in angiogenesis. Meanwhile side effects, the elevated costs and resistance problems are limiting the efficiency of mAbs as targeted therapy. The combinatory therapy with chemo or radiotherapy has improved the efficiency of mAbs. The present review aims to shed more light on the immunotherapy and the related patents that were developed for cancer treatment.

EGFR overexpression relates to triple negative profile and poor prognosis in breast cancer patients in Tunisia.

BACKGROUND: The prognosis of breast carcinoma is related to a large variety of clinical and pathological factors. Currently, only oestrogen (ER) and progesterone (PR) receptors and human epidermal growth factor receptor 2 (HER2) are used in routine pathological assessment as biomarkers. The aim of this study was to evaluate the prognostic impact of epidermal growth factor receptor (EGFR) expression individually and in combination to classical biomarkers (HER2, ER, and PR), and its relation to tumors with triple negative profile in Tunisian breast carcinoma. METHODS: Immunohistochemistry was used to estimate the rate expression of these receptors. Univariate and multivariate analyses were used to explore the prognostic significance of EGFR in this study. RESULTS: The expression rate of EGFR was 28.6%. EGFR expression was inversely correlated to that of ER (P < 0.001). Significant correlations between the expression of EGFR and the high histological Scarff-Bloom-Richardson (SBR) grade (P = 0.038) and also with tumors size (P = 0.041) were observed. The triple negative profile (TN: ER-/PR-/HER2-) was present in 17.3% of cases. EGFR overexpression was positively associated with this clinical aggressive profile (P < 0.001). Survival analysis showed that EGFR expression was associated with poor survival of patients (P = 0.004). In multivariate analysis, EGFR expression (P = 0.035) was found to be independent prognostic factors (significantly correlated to survival). CONCLUSION: EGFR overexpression was observed in 28.6% of Tunisian breast carcinoma, associated with unfavorable prognosis and with triple negative tumors. Systemically evaluation of EGFR in breast carcinoma could benefit especially to TN subgroup from EGFR targeting agents.