Rhus coriaria induces senescence and autophagic cell death in breast cancer cells through a mechanism involving p38 and ERK1/2 activation

Here, we investigated the anticancer effect of Rhus coriaria on three breast cancer cell lines. We demonstrated that Rhus coriaria ethanolic extract (RCE) inhibits the proliferation of these cell lines in a time- and concentration-dependent manner. RCE induced senescence and cell cycle arrest at G1 phase. These changes were concomitant with upregulation of p21, downregulation of cyclin D1, p27, PCNA, c-myc, phospho-RB and expression of senescence-associated β-galactosidase activity. No proliferative recovery was detected after RCE removal. Annexin V staining and PARP cleavage analysis revealed a minimal induction of apoptosis in MDA-MB-231 cells. Electron microscopy revealed the presence of autophagic vacuoles in RCE-treated cells. Interestingly, blocking autophagy by 3-methyladenine (3-MA) or chloroquine (CQ) reduced RCE-induced cell death and senescence. RCE was also found to activate p38 and ERK1/2 signaling pathways which coincided with induction of autophagy. Furthermore, we found that while both autophagy inhibitors abolished p38 phosphorylation, only CQ led to significant decrease in pERK1/2. Finally, RCE induced DNA damage and reduced mutant p53, two events that preceded autophagy. Our findings provide strong evidence that R. coriaria possesses strong anti-breast cancer activity through induction of senescence and autophagic cell death, making it a promising alternative or adjunct therapeutic candidate against breast cancer.UAEU Program for Advanced Research (Grant 31S111-UPAR) and by the Zayed Center for Health Sciences (ZCHS) research grant (grant 31R021) and College of Science Individual Research Grant (grant 31S123) to Rabah Iratni

Comparative Degradation of a Thiazole Pollutant by an Advanced Oxidation Process and an Enzymatic Approach

Organic pollutants, especially those found in water bodies, pose a direct threat to various aquatic organisms as well as humans. A variety of different remediation approaches, including chemical and biological methods, have been developed for the degradation of various organic pollutants. However, comparative mechanistic studies of pollutant degradation by these different systems are almost non-existent. In this study, the degradation of a model thiazole pollutant, thioflavin T (ThT), was carried out in the presence of either an advanced oxidation process (ultraviolet (UV) + H2O2) or a chloroperoxidase enzyme system (CPO + H2O2). The degradation was followed both spectrophotometrically and using liquid chromatography-mass spectroscopy (LC-MS), and the products formed were identified using tandem liquid chromatography-mass spectrometry-mass spectrometry (LC-MS-MS). The results show that the two remediation approaches produced different sets of intermediates, with only one common species (a demethylated form of ThT). This suggests that different degradation schemes were operating in the two systems. Interestingly, one of the major intermediates produced by the CPO + H2O2 system was a chlorinated form of thioflavin. Phytotoxicity studies showed that the CPO + H2O2-treated ThT solution was significantly (p <0.05) less toxic than the UV + H2O2-treated ThT solution. This is the first time that a comparative mechanistic study showing in detail the intermediates generated in chemical and biological remediation methods has been presented. Furthermore, the results show that different remediation systems have very different degradation schemes and result in products having different toxicities

Mitotic arrest and apoptosis in breast cancer cells induced by Origanum majorana extract: upregulation of TNF-α and downregulation of survivin and mutant p53.

BACKGROUND: In the present study, we investigated the effect of Origanum majorana ethanolic extract on the survival of the highly proliferative and invasive triple-negative p53 mutant breast cancer cell line MDA-MB-231. RESULTS: We found that O. majorana extract (OME) was able to inhibit the viability of the MDA-MB-231 cells in a time- and concentration-dependent manner. The effect of OME on cellular viability was further confirmed by the inhibition of colony growth. We showed, depending on the concentration used, that OME elicited different effects on the MDA-MB 231 cells. Concentrations of 150 and 300 µg/mL induced an accumulation of apoptotic-resistant population of cells arrested in mitotis and overexpressing the cyclin-dependent kinase inhibitor, p21 and the inhibitor of apoptosis, survivin. On the other hand, higher concentrations of OME (450 and 600 µg/mL) triggered a massive apoptosis through the extrinsic pathway, including the activation of tumor necrosis factor-α (TNF-α), caspase 8, caspase 3, and cleavage of PARP, downregulation of survivin as well as depletion of the mutant p53 in MDA-MB-231 cells. Furthermore, OME induced an upregulation of γ-H2AX, a marker of double strand DNA breaks and an overall histone H3 and H4 hyperacetylation. CONCLUSION: Our findings provide strong evidence that O. majorana may be a promising chemopreventive and therapeutic candidate against cancer especially for highly invasive triple negative p53 mutant breast cancer; thus validating its complementary and alternative medicinal use

Antipathogenic effects of structurally-related Schiff base derivatives: Structure–activity relationship

Eighteen structurally-related Schiff base derivatives, which belong to salicylidenebenzylamine and 2-hydroxy-1-naphthylidenebenzylamine families were prepared and characterized by spectroscopic techniques. All the synthesized compounds were screened in vitro for their antibacterial and antifungal activities. Human pathogenic gram-negative (Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa and Serratia marcescens), gram-positive bacteria (Staphylococcus epidermidis, Staphylococcus aureus and Bacillus subtilis), and fungi (Alternaria alternata, Aspergillus niger, Penicillium roqueforti, and Saccharomyces cerevisiae) were evaluated based on their toxicity to different concentrations of Schiff base compounds. For assessment of toxicity to pathogens, a disc diffusion assay was used to test the antimicrobial properties. The results revealed some antimicrobial activities of some of the synthesized compounds. Among the tested pathogens, the synthesized salicylidinaniline derivatives show highly potent action towards Alternaria alternata. Interestingly, a compound which contains the –Cl group is only effective against gram negative bacteria, but not gram positive bacteria. No remarkable antibacterial or antifungal activities were observed in the presence of –CH3 or –Br group. Furthermore, most of the naphthalene-containing compounds show no growth retardation towards bacterial or fungal pathogens. On the other hand, the presence of –OH or –SH group at para or ortho position, respectively, on the aniline site is correlated with an increased inhibitory drug effect on all pathogens. The importance of our findings to the drug research and developments is discussed in the context of finding a correlation between the structural properties of the new drugs and their biological activities

Development and Evaluation of Crocetin-Functionalized Pegylated Magnetite Nanoparticles for Hepatocellular Carcinoma

Liver cancer remains among the leading causes of cancer-related deaths worldwide. This is due to many reasons, including limitations of available drugs, late diagnosis due to the overlapping symptoms with many other liver diseases, and lack of effective screening modalities. Compared to conventional chemotherapy, targeted drug delivery systems are advantageous in many ways, as they minimize drug resistance and improve therapeutic value for cancer patients. Nanomaterials, in general, and nanoparticles, in particular, possess nm size, which provides a high surface area for a great extent of functionalization to be used for the targeted delivery of cancer drugs. Amongst the different formulations of nanoparticles, magnetic nanoparticles (MNPs) have unique chemical and physical characteristics and magnetic behavior, making them preferable candidates as a core for drug delivery systems. To maintain the nanosized structure of MNPs, a polymeric coating is usually applied to maintain the nanoparticles dispersed in the solution. Moreover, the polymeric coating provides a plate form for carrying drug molecules on its surface. In the present study, poly(ethylene glycol) (PEG)-coated MNPs were successfully synthesized, where the optimum concentration of PEG on the surface of the MNPs was investigated. The PEG-coated MNPs were further coated with crocetin at different concentrations. The crocetin-coated pegylated MNPs were evaluated in vitro using a hepatic cell line (HepG2) for up to 72 h. Results showed good release kinetics under acidic and neutral conditions. The optimally prepared drug delivery system showed a high potential for reducing the HepG2 cell proliferation in vitro using an MTT assay. The calculated IC50 for Cro-PEG-MNPs were 0.1019, 0.0903, and 0.0462 mg/mL of 5×, 10× and 20×, respectively

Differential regulation of survivin expression by <i>OME</i> in MDA-MB-231 cells.

<p>Western blot analysis showing a differential effect on survivin expression by different concentrations of OME in MDA-MB-231 cells. Whole cell protein were extracted from OME or vehicle (ethanol)-treated cells and subjected to Western blot analysis, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056649#s2" target="_blank">Materials and Methods</a>, for survivin and β-actin (loading control) proteins.</p

Induction of G2/M cell cycle arrest and apoptosis by <i>O. majorana</i> extract in MDA-MB-231 cells.

<p>(A) MDA-MB-231 cells (1.8×10⁶) seeded on 100 mm culture dish were exposed various concentrations of <i>O. majorana</i> extract or equal volume of vehicle (ethanol) as control for 24 h. Following treatment, cells were harvested, fixed, stained with propidium iodide, and analyzed for cell cycle distribution by flow cytometry. Data represent the mean of three independent experiments. The percentage of cells in sub-G1 (apoptosis), G1, S and G2/M appears at the upper right of each graph. (B) Expression of cell cycle regulator in OME-treated MDA-MB-231. Western blot analysis of phospho(ser10)-H3, and cyclin B1 in MDA-MB231 cells exposed for 24 h to ethanol or indicated concentrations of OME. (C) Stimulation of caspase 3/7 activity in MDA-MB-231 cells after exposure to OME (0–600 µg/mL) for 24 h and 48 h, relative to a similar amount of viable ethanol-treated cells. The relative caspase 3/7 activity was normalized to the number of viable cells per well and is expressed as fold of induction compared to the control. (D) Concentration-dependent induction of PARP cleavage in OME-treated MDA-MB231 cells. Cells were treated with or without increasing concentrations of the extract and proteins were extracted as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056649#s2" target="_blank">Materials and Methods</a>. Western blot analysis was carried out using anti-PARP antibodies. (*<i>p</i><0.05, **<i>p</i><0.005 and ***<i>p</i><0.0005).</p

Inhibition of colony growth by <i>O. majorana</i> extract.

<p>Inhibition of colony growth was assessed by measuring the size of the colonies obtained in vehicle (ethanol)- and OME-treated plates. Data were compared with those obtained for the 2 weeks colonies. Two types of colonies were counted and depending on their diameter were categorized as large (≥200 µm) and small (<200−≥50 µm).</p

<i>O. majorana</i> induces apoptosis by activation of caspase 8 and upregulation of TNF-α.

<p>(A) <i>O. majorana</i> extract induces an activation of caspase 8 but not caspase 9 in MDA-MB-231 cells. MDA-MB-231 cells were incubated with various concentrations of the extract for 24 h. The caspase activation induced by the OME was assayed as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056649#s2" target="_blank">Materials and methods</a>. The relative caspase 8 and 9 activity was normalized to the number of viable cells per well and is expressed as fold of induction compared to the control. (B) Western blot analysis showing an increase in cellular TNF-α protein in the MDA-MB-231 cells treated with OME. Whole cell protein were extracted from OME-treated cells or vehicle (ethanol)-treated cells and subjected to Western blot analysis, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056649#s2" target="_blank">Materials and Methods</a>, for TNF-α and β-actin (loading control) proteins. (C) Immunofluorescence staining for TNF-α in OME-treated MDA-MB- 231cells. Cells were treated with 150 and 300 µg/mL of the extract for 24 h, fixed, permeabilized, and then processed for immunofluorescence using antibodies against TNF-α protein. DAPI was used as a nuclear stain. (*<i>p</i><0.05 and **<i>p</i><0.005).</p