Direct and macrophage stimulation mediated effects of active, inactive, and cell-free supernatant forms of Akkermansia muciniphila and Faecalibacterium duncaniae on hepcidin gene expression in HepG2 cells.

Hepcidin is a crucial regulator of iron homeostasis with protective effects on liver fibrosis. Additionally, gut microbiota can also affect liver fibrosis and iron metabolism. Although the hepatoprotective potential of Akkermansia muciniphila and Faecalibacterium duncaniae, formerly known as F. prausnitzii, has been reported, however, their effects on hepcidin expression remain unknown. We investigated the direct and macrophage stimulation-mediated effects of active, heat-inactivated, and cell-free supernatant (CFS) forms of A. muciniphila and F. duncaniae on hepcidin expression in HepG2 cells by RT-qPCR analysis. Following stimulation of phorbol-12-myristate-13-acetate (PMA) -differentiated THP-1 cells with A. muciniphila and F. duncaniae, IL-6 concentration was assessed via ELISA. Additionally, the resulting supernatant was treated with HepG2 cells to evaluate the effect of macrophage stimulation on hepcidin gene expression. The expression of genes mediating iron absorption and export was also examined in HepG2 and Caco-2 cells via RT-qPCR. All forms of F. duncaniae increased hepcidin expression while active and heat-inactivated/CFS forms of A. muciniphila upregulated and downregulated its expression, respectively. Active, heat-inactivated, and CFS forms of A. muciniphila and F. duncaniae upregulated hepcidin expression, consistent with the elevation of IL-6 released from THP-1-stimulated cells as a macrophage stimulation effect in HepG2 cells. A. muciniphila and F. duncaniae in active, inactive, and CFS forms altered the expression of hepatocyte and intestinal iron-mediated absorption /exporter genes, namely dcytb and dmt1, and fpn in HepG2 and Caco-2 cells, respectively. In conclusion, A. muciniphila and F. duncaniae affect not only directly but also through macrophage stimulation the expression of hepcidin gene in HepG2 cells. These findings underscore the potential of A. muciniphila and F. duncaniae as a potential therapeutic target for liver fibrosis by modulating hepcidin and intestinal and hepatocyte iron metabolism mediated gene expression.

Harnessing exosomes as a platform for drug delivery in breast cancer: A systematic review for in vivo and in vitro studies.

Breast cancer remains a significant global health concern, emphasizing the critical need for effective treatment strategies, especially targeted therapies. This systematic review summarizes the findings from in vitro and in vivo studies regarding the therapeutic potential of exosomes as drug delivery platforms in the field of breast cancer treatment. A comprehensive search was conducted across bibliographic datasets, including Web of Science, PubMed, and Scopus, using relevant queries from several related published articles and the Medical Subject Headings Database. Then, all morphological, biomechanical, histopathological, and cellular-molecular outcomes were systematically collected. A total of 30 studies were identified based on the Preferred Reporting Items for Systematic Reviews and Meta-Analysis guidelines. These studies underwent assessment using the Systematic Review Centre for Laboratory Animal Experimentation risk of bias assessment tool. The results indicate that exosomes exhibit promise as effective drug delivery platforms, capable of hindering cancer cell viability, proliferation, migration, and angiogenesis. However, a comprehensive assessment is challenging due to some studies deviating from guidelines and having incomplete methodology. Addressing these, future studies should detail methodologies, optimize dosing, and enhance exosome production. Standardization in reporting, consistent protocols, and exploration of alternative sources are crucial.

Polyethylene Glycol -Mediated Exosome Isolation: A Method for Exosomal RNA Analysis.

Background: : Exosomal RNAs (ExoRNAs) offer valuable insights into their cellular origin. ExoRNA studies were faced with challenges in obtaining sufficient amounts of high-quality RNA. Herein, we aimed to compare three traditional exosome isolation methods to introduce an appropriate strategy to extract RNA from cancer-derived exosomes for further RNA analysis. Methods: Exosomes were isolated through ultracentrifugation, precipitation kit, and size exclusion column chromatography, and then characterized by dynamic light scattering and transmission electron microscopy, followed by extracting total RNA. The quality and quantity of the extracted RNAs were assessed by a NanoDrop and 2.5% agarose gel electrophoresis. Results: Extracted exosomes displayed a similar range of size and morphology. We found that polyethylene glycol-precipitation method resulted in a higher RNA yield with a 260/280 ratio of 1.9. The obtained exoRNA appeared as a smear in the agarose gel, indicative of small exoRNAs. Conclusion: We provide researchers a suitable approach to isolate exosomes based on yield and purity of exoRNA.

The effect of Bacteroides fragilis and its postbiotics on the expression of genes involved in the endocannabinoid system and intestinal epithelial integrity in Caco-2 cells.

Purpose: Gut microbiota and its derivatives by constantly interacting with the host, regulate the host function. Intestinal epithelium integrity is under the control of various factors including the endocannabinoid system (ECS). Accordingly, we aimed at investigating the effect of Bacteroides fragilis and its postbiotics (i.e., heat-inactivated, cell-free supernatants (CFS) and outer membrane vesicles (OMVs)) on the expression of genes involved in ECS (cnr1, faah, pparg) and the epithelial barrier permeability (ocln, tjp1) in a Caco-2 cell line. Methods: Caco-2 cell line was treated with live or heat-inactivated B. fragilis at MOIs of 50 and 100, or stimulated with 7% V/V CFS and B. fragilis OMVs at a dose of 50 and 100 µg/ml overnight. RT-qPCR was applied for expression analysis. Results: Heat-inactivated B. fragilis induced cnr1, pparg, tjp1, and suppressed faah expression, while live B. fragilis had the opposite effect. OMVs increased pparg, and tjp1 expression by reducing the activity of ECS through an increase in faah and a reduction in cnr1 expression. Finally, an increase in the expression of pparg and ocln, and a reduction in the expression of cnr1 was detected in Caco-2 cells treated with CFS. Conclusion: The live and heat-inactivated B. fragilis inversely affected cnr1, faah, pparg, and tjp1 expression in Caco-2 cells. Increased tjp1 mRNA levels by affecting the expression of ECS related genes is taken as an indication of the potential beneficial effects of B. fragilis postbiotics and making them potential candidates for improving permeability in the leaky gut syndrome. Supplementary Information: The online version contains supplementary material available at 10.1007/s40200-023-01264-8.

Genome-Wide Expression Analysis of Root Tips in Contrasting Rice Genotypes Revealed Novel Candidate Genes for Water Stress Adaptation.

Root system architecture (RSA) is an important agronomic trait with vital roles in plant productivity under water stress conditions. A deep and branched root system may help plants to avoid water stress by enabling them to acquire more water and nutrient resources. Nevertheless, our knowledge of the genetics and molecular control mechanisms of RSA is still relatively limited. In this study, we analyzed the transcriptome response of root tips to water stress in two well-known genotypes of rice: IR64, a high-yielding lowland genotype, which represents a drought-susceptible and shallow-rooting genotype; and Azucena, a traditional, upland, drought-tolerant and deep-rooting genotype. We collected samples from three zones (Z) of root tip: two consecutive 5 mm sections (Z1 and Z2) and the following next 10 mm section (Z3), which mainly includes meristematic and maturation regions. Our results showed that Z1 of Azucena was enriched for genes involved in cell cycle and division and root growth and development whereas in IR64 root, responses to oxidative stress were strongly enriched. While the expansion of the lateral root system was used as a strategy by both genotypes when facing water shortage, it was more pronounced in Azucena. Our results also suggested that by enhancing meristematic cell wall thickening for insulation purposes as a means of confronting stress, the sensitive IR64 genotype may have reduced its capacity for root elongation to extract water from deeper layers of the soil. Furthermore, several members of gene families such as NAC, AP2/ERF, AUX/IAA, EXPANSIN, WRKY, and MYB emerged as main players in RSA and drought adaptation. We also found that HSP and HSF gene families participated in oxidative stress inhibition in IR64 root tip. Meta-quantitative trait loci (QTL) analysis revealed that 288 differentially expressed genes were colocalized with RSA QTLs previously reported under drought and normal conditions. This finding warrants further research into their possible roles in drought adaptation. Overall, our analyses presented several major molecular differences between Azucena and IR64, which may partly explain their differential root growth responses to water stress. It appears that Azucena avoided water stress through enhancing growth and root exploration to access water, whereas IR64 might mainly rely on cell insulation to maintain water and antioxidant system to withstand stress. We identified a large number of novel RSA and drought associated candidate genes, which should encourage further exploration of their potential to enhance drought adaptation in rice.

Cyclin-dependent kinase inhibitors (CDKIs) and the DNA damage response: The link between signaling pathways and cancer.

At the cellular level, DNA repair mechanisms are crucial in maintaining both genomic integrity and stability. DNA damage appears to be a central culprit in tumor onset and progression. Cyclin-dependent kinases (CDKs) and their regulatory partners coordinate the cell cycle progression. Aberrant CDK activity has been linked to a variety of cancers through deregulation of cell-cycle control. Besides DNA damaging agents and chromosome instability (CIN), disruptions in the levels of cell cycle regulators including cyclin-dependent kinase inhibitors (CDKIs) would result in unscheduled proliferation and cell division. The INK4 and Cip/Kip (CDK interacting protein/kinase inhibitor protein) family of CDKI proteins are involved in cell cycle regulation, transcription regulation, apoptosis, and cell migration. A thorough understanding of how these CDKIs regulate the DNA damage response through multiple signaling pathways may provide an opportunity to design efficient treatment strategies to inhibit carcinogenesis.

Comparison of flagellin and an oil-emulsion adjuvant in inactivated Newcastle disease vaccine in stimulation of immunogenic parameters.

The present study was designed to investigate the potential application of native (N) and recombinant (truncated modified [tmFliC] and full-length [flFliC]) flagellin proteins along with inactivated Newcastle disease virus (NDV). Fifty six SPF chickens were immunized twice with PBS (control), inactivated NDV (Ag), inactivated NDV/flFliC (AgF), inactivated NDV/tmFliC (AgT), inactivated NDV/N (AgN), commercial vaccine containing Montanide (Vac) and Vac/N (VacN), with a two-week interval. Blood was collected weekly and spleens were harvested after chickens were sacrificed. Interleukin-6 (IL-6) and tumor necrotic factor-α (TNF-α) gene expression in peripheral blood mononuclear cells were analyzed by Real-Time PCR. Antibody response was assessed by haemagglutination inhibition (HI). Cellular activity was quantified by MTT assay. Results showed that the most IL-6 and TNF-α gene expression was observed in AgF group (P < 0.01). The lowest gene expression among vaccinated groups was observed in Ag group for IL-6 and Ag and Vac group for TNF-α. The highest HI titer was observed in Vac, VacN, AgF and AgT groups. The AgF group showed the highest cellular activity (P < 0.01). In conclusion, flagellin-adjuvanted groups showed a pro-inflammatory effect and acted similarly to or better than the Vac group. Hence, flagellin can be proposed as a potential adjuvant for ND vaccine.

Differential adaptation strategies to different levels of soil water deficit in two upland and lowland genotypes of rice: a physiological and metabolic approach.

BACKGROUND: Upland genotypes of rice are less sensitive to soil water deficit (SWD), making them suitable candidates for revealing the strategies underlying plant tolerance. The physiological factors, the biochemical traits needed to withstand oxidative stress, and the metabolite fluctuations of an upland genotype (Azucena) and an intolerant lowland genotype (IR64) genotype were measured under two levels of SWD (withholding water for 7- or 14 days) to identify SWD-responsive strategies associated with tolerance. RESULTS: After withholding water for 7 days, no significant changes in physiological and biochemical traits of Azucena were observed, whereas in IR64, significant decreases in physiological factors were recorded along with increases in oxidative-stress indicators. However, the root length of Azucena increased significantly, showing a clear stress avoidance strategy. Under a prolonged treatment (14 days), IR64 entered an oxidative-damage stage, whereas Azucena exhibited a highly efficient antioxidant system. Our metabolite analysis also revealed two different enriched pathways. After a 7-day SWD, the sugar levels were decreased in the leaves of Azucena but increased in IR64. The reduction in the sugar levels (up to 1.79-log2FC) in the Azucena leaves may be indicative of their transport to the roots, supplying the carbon source needed for root elongation. Under a 14-day treatment, proline and aspartate family members accumulated to the highest levels in Azucena, whereas an increase in the levels of aromatic amino acids with key roles in the biosynthesis of secondary metabolites was detected in IR64. CONCLUSION: The adaptation strategies identified in two types of rice genotypes in confronting SWD may assist researchers in finding the proper indicators for screening more tolerant genotypes. © 2019 Society of Chemical Industry.

Potent antitumor property of Allium bakhtiaricum extracts.

BACKGROUND: Allium species are magnificently nutritious and are commonly used as a part of the diet in Iran. They have health enhancing benefits including anticancer properties due to the presence of numerous bioactive compounds. Herein, we investigated in vitro and in vivo anticancer properties of Allium bakhtiaricum extracts. METHODS: Anti-growth activity of different fractions was explored in vitro on different cancerous cells using MTT assay, Annexin V/PI and SA-ß-gal staining, Western blotting, flowcytometric and immunofluorescence microscopic evaluations. In vivo antitumor activity was investigated in BALB/c mice bearing 4 T1 mammary carcinoma cells. RESULTS: We demonstrated that chloroformic and ethyl acetate fractions exert cytotoxic activity toward MDA-MB-231 cells, the most sensitive cell line, after 72 h of treatment with IC50 values of 0.005 and 0.006 mg/ml, respectively. Incubation of MDA-MB-231 cells with » and ½ IC50-72h concentrations of each fraction resulted in a significant G2/M cell cycle arrest. » IC50-72h concentration of the chloroform fraction led to the disruption of polymerization in mitotic microtubules. Exposure of human breast cancer cells to different concentrations of the extracts at different incubation times did not induce apoptosis, autophagy or senescence. Our in vivo study revealed that administration of the chloroform extract at a dose of 1 mg/kg/day strongly suppressed mammary tumor progression and decreased the number of proliferative cells in the lung tissues indicating its anti-metastatic effect. CONCLUSION: Our findings imply that the chloroform fraction of Allium bakhtiaricum possesses the suppressive action on breast cancer through mitotic cell cycle arrest suggesting a mechanism associated with disturbing microtubule polymerization.

Two Novel Tri-Aryl Derivatives Attenuate the Invasion-Promoting Effects of Stromal Mesenchymal Stem Cells on Breast Cancer.

BACKGROUND: The concept of Epithelial-Mesenchymal Transition (EMT) to promote carcinoma progression has been recognized as a venue for research on novel anticancer drugs. Triaryl template-based structures are one of the pivotal structural features found in a number of compounds with a wide variety of biological properties including anti-breast cancer. Among the various factors triggering EMT program, cyclooxygenase-2 (COX-2), NF-κB as well as the transforming growth factor-beta (TGF-ß) have been widely investigated. OBJECTIVE: Here, we aim to investigate the effect of two novel compounds A and B possessing triaryl structures, which interact with both COX-2 and TGF-ß active sites and suppress NF-κB activation, on EMT in a co-culture system with breast cancer and stromal cells. METHODS: MDA-MB-231 and bone-marrow mesenchymal stem (BM-MS) cells were co-cultured in a trans-well plate. Migration, matrigel-based invasion and colony formation in soft agar assays along with Real- time PCR and Western blot analysis were performed to examine the effect of compounds A and B on the invasive properties of MDA-MB-231 cells after 72 hours of co-culturing with BM-MSCs. In addition, TGF-beta interaction was investigated by Localized Surface Plasmon Resonance (LSPR). RESULTS: BM-MSCs enhanced migration, invasion and anchorage-independent growth of the co-cultured MDAMB- 231 cells. A reduction in E-cadherin level concomitant with an increase in vimentin and N-cadherin levels following the co-culture implied EMT as the underlying process. Compounds A and B inhibited invasion and anchorage-independent growth of breast cancer cells co-cultured with BM-MSCs at 10µM. The observed inhibitory effects along with an increase in E-cadherin and a reduction in vimentin and ZEB2 levels suggest that the anti-invasive properties of compounds A and B might proceed through the blockade of stromal cell-induced EMT, mediated by their interaction with TGF-beta. CONCLUSION: These findings introduce compounds A and B as novel promising agents, which prevent EMT in invasive breast cancer cells.

Agroinfiltration: a rapid and reliable method to select suitable rose cultivars for blue flower production.

Rose cultivars with blue flower color are among the most attractive breeding targets in floriculture. However, they are difficult to produce due to the low efficiency of transformation systems, interactive effects of hosts and vectors, and lengthy processes. In this study, agroinfiltration-mediated transient expression was investigated as a tool to assess the function of flower color genes and to determine appropriate host cultivars for stable transformation in Rosa hybrida. To induce delphinidin accumulation and consequently to produce blue hue, the petals of 30 rose cultivars were infiltrated with three different expression vectors namely pBIH-35S-CcF3'5'H, pBIH-35S-Del2 and pBIH-35S-Del8, harbouring different sets of flower color genes. The results obtained showed that the ectopic expression of the genes was only detected in three cultivars with dark pink petals (i.e. 'Purple power', 'High & Mora' and 'Marina') after 6-8 days. The high performance liquid chromatography analyses confirmed delphinidin accumulation in the infiltrated petals caused by transient expression of CcF3'5'H gene. Moreover, there were significant differences in the amounts of delphinidin among the three cultivars infiltrated with the three different expression vectors. More specifically, the highest delphinidin content was detected in the cultivar 'Purple power' (4.67 µg g-1 FW), infiltrated with the pBIH-35S-Del2 vector. The expression of CcF3'5'H gene in the infiltrated petals was also confirmed by real time PCR. In conclusion and based on the findings of the present study, the agroinfiltration could be regarded as a reliable method to identify suitable rose cultivars in blue rose flower production programs.

In Vitro Effects of Wistar Rat Prenatal and Postnatal Cerebrospinal Fluid on Neural Differentiation and P roliferation of Mesenchymal Stromal Cells Derived from Bone Marrow.

OBJECTIVES: Cerebrospinal fluid (CSF) plays an important role in cortical development during the fetal stages. Embryonic CSF (E-CSF) consists of numerous neurotrophic and growth factors that regulate neurogenesis, differentiation, and proliferation. Mesenchymal stem cells (MSCs) are multi-potential stem cells that can differentiate into mesenchymal and non-mesenchymal cells, including neural cells. This study evaluates the prenatal and postnatal effects of CSF on proliferation and neural differentiation of bone marrow MSCs (BM-MSCs) at gestational ages E19, E20, and the first day after birth (P1). MATERIALS AND METHODS: In this experimental study, we confirmed the mesenchymal nature of BM-MSCs according to their adherence properties and surface markers (CD44, CD73 and CD45). The multi-potential characteristics of BMMSCs were verified by assessments of the osteogenic and adipogenic potentials of these cells. Under appropriate in vitro conditions, the BM-MSCs cultures were incubated with and without additional pre- and postnatal CSF. The MTT assay was used to quantify cellular proliferation and viability. Immunocytochemistry was used to study the expression of MAP-2 and ß-III tubulin in the BM-MSCs. We used ImageJ software to measure the length of the neurites in the cultured cells. RESULTS: BM-MSCs differentiated into neuronal cell types when exposed to basic fibroblast growth factor (b-FGF). Viability and proliferation of the BM-MSCs conditioned with E19, E20, and P1 CSF increased compared to the control group. We observed significantly elevated neural differentiation of the BM-MSCS cultured in the CSF-supplemented medium from E19 compared to cultures conditioned with E20 and P1 CSF group. CONCLUSIONS: The results have confirmed that E19, E20, and P1 CSF could induce proliferation and differentiation of BM-MSCs though they are age dependent factors. The presented data support a significant, conductive role of CSF components in neuronal survival, proliferation, and differentiation.

Comparison of silicon nanoparticles and silicate treatments in fenugreek.

Silicon (Si) fertilization improves crop cultivation and is commonly added in the form of soluble silicates. However, most natural plant-available Si originates from plant formed amorphous SiO2 particles, phytoliths, similar to SiO2-nanoparticles (SiNP). In this work we, therefore, compared the effect by sodium silicate and that of SiNP on Si accumulation, activity of antioxidative stress enzymes catalase, peroxidase, superoxide dismutase, lignification of xylem cell walls and activity of phenylalanine ammonia-lyase (PAL) as well as expression of genes for the putative silicon transporter (PST), defensive (Tfgd 1) and phosphoenolpyruvate carboxykinase (PEPCK) and protein in fenugreek (Trigonella foenum-graecum L.) grown in hydroponics. The results showed that Si was taken up from both silicate and SiNP treatments and increasing sodium silicate addition increased the translocation of Si to the shoot, while this was not shown with increasing SiNP addition. The silicon transporter PST was upregulated at a greater level when sodium silicate was added compared with SiNP addition. There were no differences in effects between sodium silicate and SiNP treatments on the other parameters measured. Both treatments increased the uptake and accumulation of Si, xylem cell wall lignification, cell wall thickness, PAL activity and protein concentration in seedlings, while there was no effect on antioxidative enzyme activity. Tfgd 1 expression was strongly downregulated in leaves at Si addition. The similarity in effects by silicate and SiNP would be due to that SiNP releases silicate, which may be taken up, shown by a decrease in SiNP particle size with time in the medium.

Fetal Cerebrospinal Fluid Promotes Proliferation and Neural Differentiation of Stromal Mesenchymal Stem Cells Derived from Bone Marrow

ABSTRACT Embryonic cerebrospinal fluid (E-CSF) contains many neurotrophic and growth factors, acts as a growth medium for cortical progenitors, and can modulate proliferation and differentiation of neural stem cells. Mesenchymal stem cells (MSCs) are multipotential stem cells that can differentiate into several types of mesenchymal cells as well as nonmesenchymal cells, such as neural cells. In the present study, the effect of E-CSF on proliferation and neural differentiation of bone marrow mesenchymal stem cells (BM-MSCs) was investigated to test whether E-CSF is capable of driving these cells down the neuronal line. To verify the multipotential characteristics of BM-MSCs, the cells were analyzed for their osteogenic and adipogenic potential. Expression of the neural markers, MAP-2 and β-III tubulin, was determined by Immunocytochemistry. BM-MSCs differentiate into neuronal cell types when exposed to b-FGF. BMMSCs cells cultured in medium supplemented with CSF showed significantly elevated proliferation relative to control cells in media alone. E-CSF (E17-E19) supports viability and stimulates proliferation and, significantly, neurogenic differentiation of BM-MSCs. The data presented support an important role for CSF components, specifically neurotrophic factors, in stem cell survival, proliferation and neuronal differentiation. It is crucial to understand this control by CSF to ensure success in neural stem cell therapies.

NRAMP1 gene polymorphisms and cutaneous leishmaniasis: An evaluation on host susceptibility and treatment outcome.

BACKGROUND & OBJECTIVES: Association between polymorphisms in the natural resistance associated macrophage protein 1 (NRAMP1) gene and susceptibility to cutaneous leishmaniasis (CL) has been demonstrated worldwide; however, the reported results were inconsistent. This study aimed to determine the association of NRAMP1 variants with susceptibility to CL infection and patients' response to treatment in Isfahan province of Iran. METHODS: Peripheral blood samples were collected from 150 patients with CL and 136 healthy controls. The CL patients were treated with intralesional injection of meglumine antimoniate. The polymorphic variants at NRAMP1 (A318V and D543N) were analyzed using PCR-RFLP. The chi-square test and Fisher's exact test were used to compare frequencies of alleles and genotypes of polymorphisms between patient and healthy control populations. RESULTS: There was a statistically significant difference in the D543N (rs17235409) polymorphism between the CL patients and healthy controls (p=0.008). However, no significant association was detected for A318V (rs201565523) polymorphism between groups (p=0.26). In addition, there was a lack of association between D543N and A318V genotypes with response to treatment (p=0.54 and p=0.31, respectively). INTERPRETATION & CONCLUSION: The results indicated that genetic variations of D543N (rs17235409) might be associated with susceptibility to CL infection. These data may be used for detection of sensitive individuals and prevention of CL in endemic areas.

Anti-proliferative activity of Fumaria vaillantii extracts on different cancer cell lines.

Plant-derived natural products are known to have cancer chemo-preventive and chemo-therapeutic properties. Plant extracts or their active constituents are used as folk medicine in traditional therapies by 80% of the world population. The aim of the present study was to determine the anti-proliferative potential of Fumaria vaillantii extracts on three different cancer cell lines including malignant melanoma SKMEL-3, human breast adenocarcinoma MCF-7 and human myelogenous leukemia K562 as well as human gingival fibroblast (HGF) as normal cell line. Anti-proliferative activity was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), flowcytometry and annexin methods. Total phenolics and flavonoids were determined by Folin-Ciocalteu and aluminum chloride methods. Chloroform fraction had the lowest IC50 value at 72 h (0.1 µg/ml) in MCF-7 cells. Flowcytometry and annexin-V analysis indicated that the chloroform fraction induced necrosis in MCF-7 cells. In addition, the colorimetric methods showed that the methanolic fraction possessed the highest amount of total phenolics (33.03 ± 0.75 mg/g of dry powder) and flavonoids (10.5 ± 2.0 mg/g of dry powder). The collective data demonstrated that F. vaillantii chloroform fraction may contain effective compounds with chemo-therapeutic potential act through an apoptotic independent pathway.

Two COX-2 inhibitors induce apoptosis in human erythroleukemia K562cells by modulating NF-κB and FHC pathways.

BACKGROUND: Leukemia is distinguished by abnormal proliferation of leukocytes. Although there has been some progress in developing novel cancer therapies, no significant improvement was observed in the overall survival rate over the last decade. Selective cyclooxygenase-2 (COX-2) inhibitors are known to inhibit tumor growth by exerting antimetastatic and antiangiogenic effects through inhibition of COX -dependent and independent pathways. The ability of two new triaryl-oxadiazole derivatives, compounds A (3-(4-chlorophenyl) -5-(4-flurophenyl)-4-Phenyl-4,5-dihydro-1,2,4-oxadiazole) and B (3,5-bis(4-chlorophenyl)-4-Phenyl-4,5-dihydro-1,2,4-oxadiazole), to induce apoptosis in human erythroleukemia K562 cells was evaluated and the upstream mechanism was investigated. METHODS: K562 cells were treated with compounds A and B at their IC50 concentrations and analyzed by DAPI staining and Annexin-V-FLUOS labelling solution. Nuclear factor kappa-B (NF-κB) activation was evaluated by TransAM kit. Cyclooxygenase-2 (COX-2), Caspase-3, Bax, Bcl-2, ferritin heavy chain (FHC), extra cellular signal-regulated kinase (ERK), p-ERK and early growth response protein-1 (Egr1) levels were determined using Western blotting, while c-Myc mRNA level was investigated by RT-PCR. RESULTS: Changes in nuclear morphology and the increased annexin-V/PI staining revealed the apoptotic cell death in compounds A- and B-treated K562 cells. A significant reduction in NF-κB activity as well as FHC and p-ERK levels were detected in these cells. No change was observed in the levels of Bax, Bcl-2, Caspase-3, COX-2, c-Myc and Egr1, following treatment with the two compounds. Collectively, compounds A and B potentiate apoptosis as shown by DAPI staining, flowcytometry, FHC and p-ERK downregulation and NF-κB inactivation. CONCLUSION: Two compounds induce apoptosis in a COX-2-independent manner which also appears to be independent from mitochondria, caspase and c-Myc/Egr1 pathways.

Ethanolic extract of Ferula gummosa is cytotoxic against cancer cells by inducing apoptosis and cell cycle arrest.

Ferula gummosa Boiss. has medicinal applications in treating a wide range of diseases including cancer. The objective of this study was to evaluate the antiproliferative activities of the seed and gum extracts of F. gummosa as well as to study the effect of the potent extract on the induction of apoptosis and cell cycle arrest. Our results demonstrated that the ethanolic extract had the lowest IC50 value at 72 h (0.001 ± 1.2 mg/mL) in BHY cells. Moreover, flowcytometry and annexin-V analysis revealed that the ethanolic extract induced apoptosis and cell-cycle arrest in BHY cells at G1/S phase. In addition, colorimetric methods exhibited the highest amount of total phenolics and flavonoids in the aqueous and gum extracts (0.12 ± 0.037, 0.01 ± 2.51 mg/g of dry powder). Generally, the results obtained indicate that F. gummosa ethanol extract may contain effective compounds which can be used as a chemotherapeutic agent.

Apoptotic effects of two COX-2 inhibitors on breast adenocarcinoma cells through COX-2 independent pathway.

Recently, much effort has been directed toward the search for compounds that influence apoptosis and to understand their mechanisms of action. Cyclooxygenase (COX)-2 inhibitors may induce apoptosis through the COX-2-independent mechanism via a mitochondrial pathway. In view of the reported antiproliferative activities of two COX-2 inhibitor derivatives (1, 2) in breast cancer cells (MCF-7), the present study was undertaken to evaluate the potential of these compounds to induce apoptosis and unravel the associated mechanisms. The apoptotic activities of the two compounds were assessed using flow cytometry, fluorescence microscope, and Western blot analysis. Compounds 1 and 2-treated MCF-7 cells revealed the apoptotic cell death, as confirmed by the changes in nuclear morphology and the increased annexin-V/PI staining. Elevation of Bax to Bcl-2 ratio and activation of caspase-3 were found to be associated with the initiation of apoptosis induced by compound 1. Further investigation showed that compounds 1 and 2 inhibited NF-κB, FHC, and ERK activation, while no dramatic change was revealed in c-Myc and EGR-1 levels. Our data suggest that induction of apoptosis by compounds 1 and 2 is not associated with COX-2 expression and occurs through the NF-κB pathway, which sequentially inhibits P-ERK and FHC expression.

Morphological, biochemical and molecular characterization of twelve nitrogen-fixing bacteria and their response to various zinc concentration.

BACKGROUND: Zinc is an essential micronutrient used in the form of zinc sulfate in fertilizers in the agriculture production system. Nitrogen-fixing microorganisms are also of considerable value in promoting soil fertility. OBJECTIVES: This study aimed to investigate the degree of sensitivity to varying concentrations of zinc, in the form of ZnSO4, in different strains of Azotobacter chroococcum in a laboratory environment. MATERIALS AND METHODS: To isolate A. chroococcum strains, soil samples were collected from wheat, corn and asparagus rhizospheres and cultured in media lacking nitrogen at 30˚C for 48 hours. Strains were identified based on morphological and biochemical characteristics. The presence of the nitrogenase enzyme system was confirmed by testing for the presence of the nifH gene using PCR analysis. The minimum inhibitory concentration (MIC) and optimal zinc concentration for the growth of each strain was determined. RESULTS: A total of 12 bacterial strains were isolated from six different soil samples. A. chroococcum strains were morphologically and biochemically characterized. The presence of the nifH gene was confirmed in all the strains. MIC and the optimal zinc concentration for bacterial growth were 50 ppm and 20 ppm, respectively. CONCLUSIONS: It was concluded that increasing the concentration of zinc in the agricultural soil is harmful to beneficial microorganisms and reduces the soil fertility. A 20-ppm zinc concentration in soil is suggested to be optimal.