In-vitro study of cytotoxic and apoptotic potential of Thalassia hemprichii (Ehren.) Asch. And Enhalus acoroides (L.f.) Royle against human breast cancer cell line (MCF-7) with correlation to their chemical profile.

BACKGROUND: Breast cancer is the most common type of cancer diagnosed in women. Finding novel therapeutic agents with significant cytotoxic action and minimal adverse impact on normal cells becomes crucial. Today, natural anticancer agents present an unconventional method of treating cancer, either as a curative or preventative agent, with considerable concern for marine organisms. METHODS: The anticancer effect of the alcoholic extract of different Red Sea Seagrasses on MCF-7 human breast cancer cell line has been investigated. Seagrasses were collected from Wadi El Gamal, Red Sea and extracted. Qualitative HPLC analysis was performed on the extracts for the identification of their active biomarkers. This study was aimed to explore the cytotoxic impact of Thalassia hemprichii (Ehren.) and Enhalus acoroides (L.f.) Royle on MCF-7 and their mode of action. Their anti-proliferative effects on cancer cells were performed using Neutral red assay. On the other hand, their apoptotic effect and their capacity to induce cell cycle arrest were investigated by flow cytometry assay. The effect of Seagrasses on the mitochondrial membrane potential (ΔψM) was studied by using JC-1 mitochondrial membrane potential assay kit in Seagrasses treated cancer cells to Δψ Caspases 3/7activity was examined using the colorimetric method. Gene expression analysis and quantitative real time RT-PCR for the sea grasses on MCF-7 was performed. Immune-blotting technique for Bcl-2 and p53 was investigated. RESULTS: HPLC analysis demonstrated that the extracts contained mainly flavonoids and polyphenols such as Caffeic acid, Chlorogenic acids, catechin and kaempferol that might be responsible for these anticancer effects. Seagrasses alcoholic crude extract markedly suppressed the growth and expansion of MCF-7 cells concentration-dependently with no toxicity against normal human skin fibroblast HSF. Thalassia hemprichii and Enhalus acoroides trigger mode of cell death primarily via apoptosis as confirmed by the flow cytometry. Additionally, they have ability to induce G0/S cell cycle arrest in MCF-7. The data showed the depletion in mitochondrial membrane potential (ΔψM) in the treated cells dose-dependently Caspases 3/7activities markedly increased following 24 h treatment. Finally, Gene expression analysis showed a marked reduction in Bcl-2, Survivin and CDC2 gene expression levels and a significant increase in the expression of p53 and CC2D1A as compared to control cells. CONCLUSION: In summary, the Methanolic extract of seagrass, Thalassia hemperchii and Enhalus ocoroides are able to induce concentration-dependent cytotoxic effects in human MCF-7 cells through intrinsic pathway of apoptosis in MCF-7 cells. This study reveals the beneficial importance of sea grasses as a source of anticancer agents. Further in vivo study is recommended for the active isolated biomolecules.

Biosynthesis of Zinc Oxide Nanoparticles Using Bacillus paramycoides for In Vitro Biological Activities and In Vivo Assessment Against Hepatorenal Injury Induced by CCl4 in Rats.

Recently, impressive developments in the field of nanotechnology have been achieved. The study aimed to synthetize zinc oxide nanoparticles (ZnONPs) from locally isolated terrestrial Bacillus paramycoides (MCCC 1A04098) bacteria and assess its role as antioxidant, antimicrobial, and anticancer agent. The antioxidant activity was done using the percentage of DPPH scavenging method. The antibacterial activity was evaluated against Escherichia coli, Staphylococcus aureus, Bacillus cereus, and Candida albicans. The anti-proliferation assay against hepatocellular carcinoma (HepG2) and human breast cancer (MCF-7) cell lines was estimated by neutral red assay. The apoptotic effect of ZnONP was measured by flow cytometry. The in vivo evaluation was carried out against hepatorenal injuries induced by carbon tetrachloride (CCl4) in rats comparing with silymarin as a reference drug. The oxidative stress markers, liver and kidney function enzyme indices, lipid profile, and the histological features of the liver and kidney were also examined. ZnONPs revealed antioxidant and antibacterial effects. It also exerted cytotoxic and apoptotic effect in a dose dependent manner without any toxicity on normal cell line. ZnONPs improved all the biochemical parameters under investigation to varying degrees, and the histological pictures of the liver and kidney confirmed the results. In conclusion, ZnONPs were successfully synthesized from the terrestrial Bacillus paramycoides and recorded in vitro antioxidant, anticancer, and antibacterial effects as well as in vivo anti-hepatorenal toxicity effects.

Restoring microRNA-34a overcomes acquired drug resistance and disease progression in human breast cancer cell lines via suppressing the ABCC1 gene.

PURPOSE: Breast cancer is one of the leading types of cancer diagnosed in women. Despite the improvements in chemotherapeutic cure strategies, drug resistance is still an obstacle leading to disease aggressiveness. The small non-coding RNA molecules, miRNAs, have been implicated recently to be involved as regulators of gene expression through the silencing of mRNA targets that contributed to several cellular processes related to cancer metastasis. Hence, the present study aimed to investigate the beneficial role and mechanism of miRNA-34a-based gene therapy as a novel approach for conquering drug resistance mediated by ATP-binding cassette (ABC) transporters in breast cancer cells, besides exploring the associated invasive behaviors. MATERIAL AND METHODS: Bioinformatics tools were used to predict miRNA ABC transporter targets by tracking the ABC transporter pathway. After the establishment of drug-resistant breast cancer MCF-7 and MDA-MB-231 sublines, cells were transfected with the mimic or inhibitor of miRNA-34a-5p. The quantitative expression of genes involved in drug resistance was performed by QRT-PCR, and the exact ABC transporter target specification interaction was confirmed by dual-luciferase reporter assay. Furthermore, flow cytometric analysis was utilized to determine the ability of miRNA-34a-treated cells against doxorubicin uptake and accumulation in cell cycle phases. The spreading capability was examined by colony formation, migration, and wound healing assays. The apoptotic activity was estimated as well. RESULTS: Our findings firstly discovered the mechanism of miRNA-34a-5p restoration as an anti-drug-resistant molecule that highly significantly attenuates the expression of ABCC1 via the direct targeting of its 3'- untranslated regions in resistant breast cancer cell lines, with a significant increase of doxorubicin influx by MDA-MB-231/Dox-resistant cells. Additionally, the current data validated a significant reduction of metastatic potentials upon miRNA-34a-5p upregulation in both types of breast cancer-resistant cells. CONCLUSION: The ectopic expression of miRNA-34a ameliorates the acquired drug resistance and the migration properties that may eventually lead to improved clinical strategies and outcomes for breast cancer patients. Additionally, miRNA-34a could be monitored as a diagnostic/prognostic biomarker for resistant conditions.

Neuroprotective effects of vitamin D in an Alzheimer's disease rat model: Improvement of mitochondrial dysfunction via calcium/calmodulin-dependent protein kinase kinase 2 activation of Sirtuin1 phosphorylation.

Mitochondrial dysfunction is an early event in Alzheimer's disease (AD) pathogenesis. To assess the impact of vitamin D3 (Vit.D) on neurogenesis, we investigated its role in mitigating cognitive impairment and mitochondrial dysfunction through calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2)-mediated phosphorylation of Sirtuin1 (SIRT1) in an aluminum-chloride-D-galactose (AlCl3-D-gal)-induced AD rat model. Rats were distributed into four groups: control, AlCl3 + D-gal (10 + 60 mg/kg, ip), Vit.D (500 IU/kg, po), and AlCl3 + D-gal+Vit.D. Novel object recognition (NOR), Morris Water Maze, and passive avoidance (PA) tests were used to measure memory abilities. The hippocampal tissue was used to assess vitamin D3 receptor (VDR) and peroxisome-proliferator-activated-receptor-γ-coactivator-1α (PGC-1α) expression by quantitative real-time polymerase chain reaction (qRT-PCR), CAMKK2, p-SIRT1, phosphorylated-AMP-activated protein kinase (p-AMPK), dynamin-related-protein-1 (Drp1), and mitofusin-1 (Mnf1) proteins by western blot and Ca2+ levels, endothelial nitic oxide synthase (eNOS), superoxide dismutase (SOD), amyloid beta (Aß), and phospho tau (p-Tau) via enzyme-linked immunosorbent assay(ELISA) in addition to histological and ultrastructural examination of rat's brain tissue. Vit.D-attenuated hippocampal injury reversed the cognitive decline and Aß aggregation, and elevated p-Tau levels in the AlCl3 + D-gal-induced AD rat model. In AlCl3 + D-gal-exposed rats, Vit.D induced VDR expression, normalized Ca2+ levels, elevated CAMKK2, p-AMPK, p-SIRT1, and PGC-1α expression. Vit.D reduced Drp1, induced Mnf1, increased mitochondrial membrane potential, preserved mitochondrial structure, restored normal mitochondrial function, and retained normal eNOS level and SOD activity in AlCl3 + D-gal rats. In conclusion, our findings proved that Vit.D may ameliorate cognitive deficits in AlCl3 + D-gal-induced AD by restoring normal mitochondrial function and reducing inflammatory and oxidative stress via CAMKK2-AMPK/SIRT1 pathway upregulation.

Dysregulated miRNA-375, IL-17, TGF-ß, and Microminerals Are Associated with Calpain-10 SNP 19 in Diabetic Patients: Correlation with Diabetic Nephropathy Stages.

Zinc (Zn) and copper (Cu) have been shown to have the potential to improve glucose metabolism through interactions with cytokines and signaling events with multiple genes. miRNA-375 and the Calpin-10 gene are potential genetic biomarkers for the early prediction of diabetic nephropathy (DN). 128 healthy controls and 129 type 2 diabetic (T2DM) participants were matched for age and sex. Three subgroups were identified from the T2DM group: 39 patients had microalbuminuria, 41 had macroalbuminuria, and 49 patients had renal problems. Circulating miR-375 expression levels were measured via qPCR. Calpain-10 SNP 19 (rs3842570) genotyping was assessed with allele-specific PCR in all the included participants. Spectrophotometry was used to measure the concentrations of serum copper, zinc, and magnesium, while ELISA was used to measure the levels of TGF-ß and IL-17. There was significant up-regulation in the expression of miR-375 and serum levels of TGF-ß, IL-17, Cu, and the Cu/Zn ratio, whereas, in contrast to the control group, the Zn and Mg levels were lower in the T2DM group. The DN groups had significantly lower miR-375, TGF-ß, IL-17, Mg, and Zn levels compared with the T2DM without nephropathy group. Furthermore, between TGF-ß, IL-17, and miRNA-375, there were notable correlations. Calpain-10 SNP 19 genotype 22 and allele 2 were linked to a higher incidence of T2DM and DN. Significant TGF-ß, Cu, Cu/Zn ratio, HbAc1, and creatinine levels, but insignificant miRNA-375 levels, were associated with genotype 22 of Calpain-10 SNP 19. interactions between the Calpain-10 SNP 19 genotype 22 and IL-17, TGF-ß, mineral levels, and miRNA-375 might contribute to the aetiology of DN and T2DM and may have clinical implications for diagnosis and management.

Impact of atorvastatin and mesenchymal stem cells combined with ivermectin on murine trichinellosis.

Trichinellosis is one of the global food-borne parasitic diseases that can cause severe tissue damage. The traditionally used drugs for the treatment of trichinellosis have limited efficacy against the encysted larvae in the muscular phase of the disease. Therefore, this study aimed to evaluate the role of atorvastatin and mesenchymal stem cells combined with ivermectin against different phases of Trichinella in experimentally infected mice. A total of 120 male Swiss albino mice were divided into two major groups (n = 60 of each), intestinal and muscular phases. Then, each group was subdivided into 10 subgroups (n = 6); non-infected control, infected non-treated control, infected ivermectin treated, infected atorvastatin treated, infected mesenchymal stem cells treated, infected combined ivermectin and atorvastatin treated, infected combined mesenchymal stem cells and ivermectin treated, infected combined mesenchymal stem cells and atorvastatin treated, infected combined mesenchymal stem cells and a full dose of (ivermectin and atorvastatin) treated, and infected combined mesenchymal stem cells and half dose of (ivermectin and atorvastatin) treated. Mice were sacrificed at days 5 and 35 post-infection for the intestinal and muscular phases, respectively. The assessment was performed through many parameters, including counting the adult intestinal worms and muscular encysted larvae, besides histopathological examination of the underlying tissues. Moreover, a biochemical assay for the inflammatory and oxidative stress marker levels was conducted. In addition, levels of immunohistochemical CD31 and VEGF gene expression as markers of angiogenesis during the muscular phase were investigated. The combined mesenchymal stem cells and atorvastatin added to ivermectin showed the highest significant reduction in adult worms and encysted larvae counts, the most noticeable improvement of the histopathological changes, the most potent anti-inflammatory (lowest level of IL-17) and anti-angiogenic (lowest expression of CD31 and VEGF) activities, and also revealed the highly effective one to relieve the oxidative stress (lowest level of SOD, GSH, and lipid peroxidase enzymes). These observed outcomes indicate that adding mesenchymal stem cells and atorvastatin to ivermectin synergistically potentiates its therapeutic efficacy and provides a promising candidate against trichinellosis.

Sertraline Pre-Treatment Attenuates Hemorrhagic Transformation Induced in Rats after Cerebral Ischemia Reperfusion via Down Regulation of Neuronal CD163: Involvement of M1/M2 Polarization Interchange and Inhibiting Autophagy.

Cerebral ischemia reperfusion (I/R) is one of the neurovascular diseases which leads to severe brain deterioration. Haemorrhagic transformation (HT) is the main complication of ischemic stroke. It exacerbates by reperfusion, causing a more deleterious effect on the brain and death. The current study explored the protective effect of sertraline (Sert) against cerebral I/R in rats by inhibiting HT, together with the molecular pathways involved in this effect. Forty-eight wister male rats were divided into 4 groups: Sham, Sert + Sham, I/R, and Sert + I/R. The ischemic model was induced by bilateral occlusion of the common carotid artery for 20 min, then reperfusion for 24 h. Sertraline (20 mg/kg, p.o.) was administrated for 14 days before exposure to ischemia. Pre-treatment with Sert led to a significant attenuation of oxidative stress and inflammation. In addition, Sert attenuated phosphorylation of extracellular regulated kinases and nuclear factor kappa-p65 expression, consequently modulating microglial polarisation to M2 phenotype. Moreover, Sert prevented the hemorrhagic transformation of ischemic stroke as indicated by the notable decrease in neuronal expression of CD163, activity of Heme oxygenase-2 and matrix metalloproteinase-2 and 9 levels. In the same context, Sert decreased levels of autophagy and apoptotic markers. Furthermore, histological examination, Toluidine blue, and Prussian blue stain aligned with the results. In conclusion, Sert protected against cerebral I/R damage by attenuating oxidative stress, inflammation, autophagy, and apoptotic process. It is worth mentioning that our study was the first to show that Sert inhibited hemorrhagic transformation. The protective effect of sertraline against injury induced by cerebral ischemia reperfusion via inhibiting Hemorrhagic transformation.

Linagliptin and secoisolariciresinol diglucoside attenuate hyperlipidemia and cardiac hypertrophy induced by a high-methionine diet in rats via suppression of hyperhomocysteinemia-induced endoplasmic reticulum stress.

Background: Cardiac hypertrophy (CH) is one of the contributing causes of morbidity and mortality. Hyperhomocysteinemia (HHcy) is one of the diseases which may predispose hyperlipidemia and CH. Linagliptin (Lina) and secoisolariciresinol diglucoside (SDG) are known to alleviate a variety of illnesses by reducing oxidative stress and inflammation. Aim: This study aimed to study the effect of HHcy on cardiac tissues, with a special focus on endoplasmic reticulum (ER) stress as a mainstay pathophysiological pathway. In addition, our study examined the protective effect of Lina, SDG, and their combination against HHcy-induced hyperlipidemia and CH in rats. Methods: Seventy-five male Sprague-Dawley rats were randomly divided into five groups, and for 60 days, the following regimen was administered: Group I: rats received distilled water; Group II: rats received methionine (MET) (2 g/kg/day, p.o.); groups III and IV: rats received Lina (3 mg/kg/day, p.o.) and SDG (20 mg/kg/day, p.o.), respectively, followed by MET (2 g/kg/day, p.o.); Group V: rats received Lina and SDG, followed by MET (2 g/kg/day, p.o.). Results: Pretreatment with Lina, SDG, and their combination showed a significant decrease in serum levels of HHcy and an improved lipid profile compared to the MET group. Moreover, both drugs improved cardiac injury, as evidenced by the substantial improvement in ECG parameters, morphological features of the cardiac muscle, and reduced serum levels of cardiac markers. Additionally, Lina and SDG significantly attenuated cardiac oxidative stress, inflammation, and apoptosis. Furthermore, Lina, SDG, and their combination remarkably downregulated the enhanced expression of endoplasmic reticulum (ER) stress markers, GRP78, PERK, ATF-4, CHOP, NF-κB, and SREBP1c compared to the MET-group. Conclusion: Lina and SDG showed cardioprotective effects against HHcy-induced heart hypertrophy and hyperlipidemia in rats.

Targeting the adenosine monophosphate-activated protein kinase signalling pathway by bempedoic acid attenuates Angiotensin II-induced cardiac remodelling in renovascular hypertension in rats.

The crosstalk between the renin-angiotensin system and Adenosine monophosphate-activated protein kinase (AMPK) gained significant interest due to their involvement in the pathogenesis of several cardiovascular diseases. Angiotensin II (Ang II) plays a crucial role in developing cardiac remodelling by inducing energy imbalance, inflammation, oxidative and endoplasmic reticulum stress, and transforming growth factor-ß (TGF-ß)-induced fibrosis. Ang II directly or through extracellular signal-regulated kinase (ERK) activation impairs AMPK signalling with well-known antioxidant, anti-inflammatory, and anti-fibrotic effects. AIM: This study aimed to investigate the role of bempedoic acid, a novel antihyperlipidemic drug, in attenuating hypertension-induced cardiac remodelling in rats by modulating Ang II-induced damage and activating the AMPK signalling pathway. METHOD: Sixty adult male Sprague Dawley rats were randomly allocated into the Sham control group, Hypertensive group, Captopril group (30 mg/kg), and Bempedoic acid group (30 mg/kg). Hypertension was induced by left renal artery ligation in all groups except the Sham control group. Treatment with captopril and bempedoic acid started 14 days post-surgy and lasted two weeks. Finally, Hemodynamic measurements and electrocardiographic examination were done followed by heart tissue samples collection for biochemical, histopathological, and immunohistochemical examinations. KEY FINDINGS: Bempedoic acid preserved the cardiac function and electrocardiogram patterns. It inhibited endoplasmic reticulum stress, exhibited antioxidant activity, and increased endothelial nitric oxide synthase activity. Bempedoic acid interfered with ERK signalling pathways, including nuclear factor-κB and TGF-ß, exerting anti-inflammatory and anti-fibrotic effects. SIGNIFICANCE: These findings indicate the cardioprotective and antihypertrophic activity of bempedoic acid, which are suggested to result from energy-independent AMPK downstream signalling activation.

Effect of bempedoic acid on angiotensin-II induced hypertension and vascular tissue remodelling in renal hypertensive rats through AMPK multiple signalling pathways modulation.

Angiotensin II (Ang II), the effector of the renin-angiotensin system (RAS), is a key player in the pathogenesis of chronic hypertension, accompanied by vascular tissue resistance, remodelling, and damage. Chronic activation of Ang II receptor 1 (AT-1R) impairs multiple cellular targets implicated in cellular protection and survival, including adenosine Monophosphate-activated protein kinase (AMPK) signalling. In addition, it induces oxidative damage, endoplasmic reticulum (ER) stress, and fibrotic changes in resistance vessels. Our study investigated the antihypertensive and antifibrotic effects of bempedoic acid, a first-in-class antihyperlipidemic drug that targets adenosine triphosphate-citrate lyase enzyme to inhibit cholesterol synthesis. We also studied the modulation of multiple AMPK signalling pathways by bempedoic acid in a chronic hypertension model in rats. Sixty male Sprague-Dawley rats were divided into four groups: sham group, hypertensive group, standard captopril group, and bempedoic treated group. All groups underwent left renal artery ligation except the sham group. Fourteen days post-surgery, captopril and bempedoic acid were administered with a dose of 30 mg/kg/day orally to captopril-standard and bempedoic acid-treated groups for two weeks, respectively. In mesenteric resistance arteries, bempedoic acid activated AMPK energy independently and augmented AMPK multiple cellular targets to adapt to Ang II-induced cellular stress. It exerted antioxidant activity, increased endothelial nitric oxide synthase, and reversed the ER stress. Bempedoic acid maintained vascular integrity and prevented vascular remodelling by inhibiting extracellular signal-regulated kinase (ERK)/transforming growth factor-ß fibrotic pathway. These effects were reflected in the improved hemodynamic measurements.

Potentiating the Cytotoxic Activity of a Novel Simvastatin-Loaded Cubosome against Breast Cancer Cells: Insights on Dual Cell Death via Ferroptosis and Apoptosis.

PURPOSE: Female breast cancer is the most prevalent cancer worldwide. Emerging evidence shows that simvastatin (SIM) has promising anticancer activities. However, the underlying mechanisms are not fully elucidated. Increasing reports imply statins can modulate ferroptosis through disrupting reactive oxygen species (ROS) and glutathione peroxidase enzyme (GPX4) levels. However, whether ferroptosis contributes to SIM anticancer activity, especially regarding GPX4 is unclear. Moreover, poor aqueous SIM solubility hinders its delivery in adequate levels to tumor sites. Meanwhile, cubosomes are biocompatible nanocarriers that enhance lipophilic drug delivery. Therefore, in this study, we formulated a novel SIM-loaded cubosome (SIM-CB) and analyzed its cytotoxic activity on MCF-7 cancer cells in comparison with free SIM. METHODS: The present study tested the cytotoxic activity of SIM-CB on MCF-7 cells, in comparison with SIM using sulphorhodamine assay. We analyzed SIM-CB effect on apoptosis and cell cycle using flowcytometry, and investigated its effect on Bcl-2, caspase 3, ROS, reduced glutathione (GSH), lipid peroxides and GPX4 enzyme. Finally, we tested the persistence of SIM-CB apoptosis and ferroptosis activities on MCF-7 cells in presence of vitamin E, a potent antioxidant and ferroptosis inhibitor. RESULTS: SIM-CB was successfully formulated at the nano size. SIM-CB significantly increased simvastatin therapeutic activity, with IC50 of SIM-CB 52% lower than SIM. 95% CI [1.8, 2.7], SD = 0.34 for SIM-CB, and [4.1, 5.2], SD = 0.45 for SIM. Compared with free SIM, SIM-CB doubled total deaths and increased apoptosis (p < 0.05). Moreover, SIM-CB remarkably increased caspase-3, ROS, and lipid peroxide levels but decreased antiapoptotic Bcl-2 protein, GSH, and GPX4 compared with free SIM. Notably, SIM-CB elicited a high distinguished resistance against the inhibitory effects of vitamin E. CONCLUSION: To the best of our knowledge, this study is the first to present SIM-CB as a promising means to enhancing the therapeutic potential of simvastatin against breast cancer cells, through potentiating both apoptosis and ferroptosis.

COVID-19 Vaccination Perception and Attitude among Healthcare Workers in Egypt.

INTRODUCTION: COVID-19 pandemic has affected the whole world, especially the frontline worriers. To get shielded through this war, the world is racing to reach and manufacture COVID-19 vaccines. Vaccination hesitancy is one of the significant obstacles to global health. OBJECTIVES: This study aimed to assess the perception and attitude of healthcare workers in Egypt toward COVID-19 vaccines, acknowledge the determinants of their attitude, and the factors that could increase the acceptance of the vaccine. METHODS: an observational web-based anonymous survey was conducted on 385 Egyptian healthcare workers in different governorates. The questionnaire-based on Vaccine Hesitancy Survey Questions of the World Health Organization was available in Arabic and English languages and was tested for reliability. RESULTS: Regarding vaccination decision, 51% of the participants were undecided, 28% refused, and 21% accepted vaccination. Reasons for vaccine acceptance mainly were risks of COVID-19 (93%), safety (57.5%), and effectiveness (56.25%) of the vaccine. Simultaneously, the reasons for vaccine hesitancy were the absence of enough clinical trials (92.4%) and fear of side effects of the vaccine (91.4%). The leading factor that could increase vaccination acceptance among the participants was to get sufficient and accurate information about the available vaccines. The participants revealed a high mean level of concern for COVID-19 vaccines' safety (3.8 of 5) that differs significantly among the different study groups (P-value .002). CONCLUSION: Despite the COVID-19 pandemic, only approximately 21% of Egyptian healthcare workers in our study accepted the COVID-19 vaccination. Vaccine hesitancy represents a major barrier to implementing vaccination programs.

Lignans and Polyphenols of Phyllanthus amarus Schumach and Thonn Induce Apoptosis in HCT116 Human Colon Cancer Cells through Caspases-Dependent Pathway.

BACKGROUND: The anticancer effects of Phyllanthus amarus extract on various cancer cells have been investigated, however, the effects of its major constituents on HCT116 human colorectal cancer cells have not been reported. OBJECTIVE: In the present study, we investigated the cytotoxic effect of 80% ethanol extract of P. amarus and its marker constituents (phyllanthin, hypophyllanthin, gallic acid, niranthin, greraniin, phyltetralin, isolintetralin, corilagin and ellagic acid) on HCT116 and their underlying mechanisms of action. METHODS: Their antiproliferative and apoptotic effects on HCT 116 were performed using MTT assay and flow cytometric analysis, respectively, while caspases 3/7, 8 and 9 activities were examined using the colorimetric method. The expression of cleaved poly ADP ribose polymerase enzyme (PARP) and cytochrome c proteins was investigated by the immune-blot technique. RESULTS AND DISCUSSION: HPLC and LC-MS/MS analyses demonstrated that the extract contained mainly lignans and polyphenols. The plant samples markedly suppressed the growth and expansion of HCT116 cells in a concentration- and time-dependent manner with no toxicity against normal human fibroblast CCD18 Co. P. amarus extract, phyllanthin and gallic acid induced mode of cell death primarily through apoptosis as confirmed by the exteriorization of phosphatidylserine. Caspases 3/7, 8, and 9 activities increased in a concentration-dependent manner following 24h treatment. The expressions of cleaved PARP (Asp 214) and cytochrome c were markedly upregulated. CONCLUSION: P. amarus extract, phyllanthin and gallic acid exhibited an apoptotic effect on HCT116 cells through the caspases-dependent pathway.

Luteolin protects against testicular injury induced by lead acetate by activating the Nrf2/HO-1 pathway.

Lead (Pb) is a toxic heavy metal that is harmful to humans, especially male reproductive organs. Luteolin (LUT) is a naturally occurring flavonoid with numerous biological activities. Our aim was to investigate the possible reproprotective effect of LUT against testicular deficits induced by Pb intoxication. In the present study, 28 rats were distributed into 4 groups: control, LUT (50 mg/kg), lead acetate (PbAc, 20 mg/kg), and LUT + PbAc groups, in which rats were pre-treated with LUT 3 hr before PbAc injection. All animals were treated for 7 days. Oxidative stress, inflammatory and apoptotic markers along with histopathological changes have been examined using spectrophotometric, ELISA, real-time PCR, and histopathological methods. PbAc injection elevated Pb concentration in testicular tissue and decreased levels of sex hormones. PbAc intoxication exacerbated lipoperoxidation and nitric oxide formation, depleted superoxide dismutase, and catalase activities along with glutathione and its originated enzymes (glutathione peroxidase and glutathione reductase). At the molecular level, PbAc deactivated nuclear factor erythroid 2-related factor 2 and heme oxygenase-1 in the testicular tissue. In addition, PbAc toxicity induced inflammatory and apoptotic cascades in testicular tissue as evidenced by the increased tumor necrosis factor-alpha, interleukin-1 beta, inducible nitric oxide synthase, Bax, and caspase 3, while Bcl-2 was declined. Histopathological examination of testicular tissue also revealed that PbAc caused degeneration alterations in spermatogenic cells, the spermatogenic epithelial cells were disconnected from the basement membrane, and the seminiferous tubules were vacuolated. Remarkably, pre-treatment with LUT minimized significantly the testicular damage induced by PbAc. Therefore, we conclude that LUT may have a beneficial effect against PbAc-induced testicular injury through preventing oxidative challenge, inflammation, and finally apoptosis.

Synthesis of Graphene Oxide Interspersed in Hexagonal WO3 Nanorods for High-Efficiency Visible-Light Driven Photocatalysis and NH3 Gas Sensing.

WO3 nanorods and GO (at 1 wt% loading) doped WO3 were synthesized using a template free deposition-hydrothermal route and thoroughly characterized by various techniques including XRD, FTIR, Raman, TEM-SAED, PL, UV-Vis, XPS, and N2 adsorption. The nano-materials performance was investigated toward photocatalytic degradation of methylene blue dye (20 ppm) under visible light illumination (160 W, λ> 420) and gas sensing ability for ammonia gas (10-100 ppm) at 200°C. HRTEM investigation of the 1%GO.WO3 composite revealed WO3 nanorods of a major d-spacing value of 0.16 nm indexed to the crystal plane (221). That relevant plane was absent in pure WO3 establishing the intercalation with GO. The MB degradation activity was considerably enhanced over the 1%GO.WO3 catalyst with a rate constant of 0.0154 min-1 exceeding that of WO3 by 15 times. The reaction mechanism was justified dependent on electrons, holes and •OH reactive species as determined via scavenger examination tests and characterization techniques. The drop in both band gap (2.49 eV) and PL intensity was the main reason responsible for enhancing the photo-degradation activity of the 1%GO.WO3 catalyst. The later catalyst initiated the two electron O2 reduction forming H2O2, that contributed in the photoactivity improvement via forming •OH moieties. The hexagonal structure of 1%GO.WO3 showed a better gas sensing performance for ammonia gas at 100 ppm (Ra-Rg/Rg = 17.6) exceeding that of pure WO3 nanorods (1.27). The superiority of the gas-sensing property of the 1%GO.WO3 catalyst was mainly ascribed to the high dispersity of GO onto WO3 surfaces by which different carbon species served as mediators to hinder the recombination rate of photo-generated electron-hole pairs and therefore facilitated the electron transition. The dominancy of the lattice plane (221) in 1%GO.WO3 formed between GO and WO3 improved the electron transport in the gas-sensing process.

ERK activation by zeranol has neuroprotective effect in cerebral ischemia reperfusion.

AIMS: Incidence of stroke increases in postmenopausal women with dangerous consequences. In this study we used zeranol to protect ovariectomized (OVX) rats against cerebral I/R damage and our target is to identify the mechanism of its protection, in addition to investigating whether this mechanism inhibits inflammation (by preventing glial cell activation) and apoptosis. MAIN METHODS: First 18 ovariectomized rats were allocated into 3 groups: I/R group, zeranol+ I/R group and U0126, MEK1/2 inhibitor + zeranol+ I/R group. After 24 h reperfusion, protein expression of total extracellular signal-regulated protein kinase (t-ERK1/2), phosphorylated extracellular signal-regulated protein kinase (p-ERK1/2), Bcl-2, and Bax were quantified. Second 36 female rats were allocated into 3 groups: sham group, I/R group (after ovariectomy by 7 weeks, rats exposed to cerebral I/R) and zeranol group (after ovariectomy by 2 weeks, rats received zeranol for 5 weeks). After 24 h of reperfusion, the following parameters were measured; total nitrate/nitrite, interleukin-10, myeloperoxidase, caspase-3, and finally immunohistochemistry analysis of glial fibrillary acidic protein, cyclooxygenase-2 in cortex and hippocampus (CA1) regions were performed. KEY FINDINGS: U-0126 administration reversed the neuroprotective effect induced by zeranol through decreasing ratio of p-ERK1/2:ERK1/2 and Bcl-2/Bax in brain tissue. Activation of ERK signaling pathway by zeranol caused reduction in brain apoptosis and inflammation. SIGNIFICANCE: Zeranol showed protective effect in OVX rats that were exposed to cerebral I/R by activation of ERK signaling pathway which was blocked by U0126. This protective effect in turns led to decrease inflammation and apoptosis.

The protective effect of zeranol in cerebral ischemia reperfusion via p-CREB overexpression.

AIMS: Cerebral ischemia reperfusion (I/R) is a neurovascular disease leading to cerebral damage. It was found that postmenopausal women are liable to more dangerous effects than men at same age in stroke. The objective of this study is to investigate the neuroprotective effect of zeranol against cerebral ischemia reperfusion in ovariectomized rats. MAIN METHODS: 36 female wistar rats divided in to 3 groups: sham group, I/R group (where I/R was induced 7 weeks after ovariectomy), zeranol group (0.5 mg/kg every 3 days for 5 weeks before I/R). Cerebral ischemia reperfusion (I/R) was performed by bilateral common carotid artery occlusion then de-ligated to restore blood flow. After 24 h of reperfusion, rats performed cylinder test to evaluate behavioral dysfunction followed by decapitation. Brain tissues were collected for biochemical measures such as oxidative stress marker malondialdehyde, antioxidant markers reduced glutathione, inflammatory markers (interleukin-1 beta, tumor necrosis factor alpha, and inducible nitric oxide synthase), matrix metalloproteinase-9, adenosine triphosphate, brain derived neurotrophic factor, glucose transporter-3, phosphorylated c-AMP response element binding protein and finally nissl staining for histopathological examination. KEY FINDINGS: The zeranol administered group showed a reversal of neuronal damage caused by ischemia evidenced by the decrease in MDA, IL-1ß, TNF-α, and MMP-9 levels, increase GSH, and ATP levels, decrease expression of iNOS in both regions cortex and hippocampus, increase protein level of p-CREB, GLUT-3 and BDNF, increase number of intact neuron cells in both regions and attenuated histological changes in both cortex and hippocampus regions. SIGNIFICANCE: Zeranol has neuroprotective potential against cerebral ischemia reperfusion in ovariectomized rats.

Dendritic cells pulsed with generated tumor cell lysate from Phyllanthus amarus Schum. & Thonn. induces anti-tumor immune response.

BACKGROUND: Dendritic cells (DCs) are unique antigen presenting cells (APC) which play a pivotal role in immunotherapy and induction of an effective immune response against tumors. In the present study, 80% ethanol extract of Phyllanthus amarus was used to generate tumor lysate (TLY) derived from HCT 116 and MCF-7 cancer cell lines via induction of apoptosis. Monocyte-derived DCs were generated ex vivo from the adherent population of peripheral blood mononuclear cells (PBMCs). The generated TLY were used to impulse DCs to investigate its effect on their cellular immune functions including antigen presentation capacity, phagocytic activity, chemotaxis capacity, T-cell proliferation and cytokines release. METHODS: The effect of P. amarus-generated TLY on DCs maturation was evaluated by determination of MHC class I, II and CD 11c expression as well as the co-stimulatory molecules CD 83 and 86 by using flow cytometry. The phagocytic capacity of TLY-pulsed DCs was investigated through FITC-dextran uptake by using flow cytometry. The effect on the cytokines release including IL-12, IL-6 and IL-10 was elucidated by using ELISA. The migration capacity and T cell proliferation activity of pulsed DCs were measured. The relative gene expression levels of cytokines were determined by using qRT-PCR. The major constituents of P. amarus extract were qualitatively and quantitatively analyzed by using validated reversed-phase high performance liquid chromatography (HPLC) methods. RESULTS: P. amarus-generated TLY significantly up-regulated the expression levels of MHC class I, CD 11 c, CD 83 and 86 in pulsed DCs. The release of interleukin IL-12 and IL-6 was enhanced by TLY-DCs at a ratio of 1 DC: 3 tumor apoptotic bodies (APO), however, the release of IL-10 was suppressed. The migration ability as well as allogeneic T-cell proliferation activities of loaded DCs were significantly enhanced, but their phagocytic capacity was highly attenuated. The gene expression profiles for IL-12 and IL-6 of DCs showed increase in their mRNA gene expression in TLY pulsed DCs versus unloaded and LPS-treated only DCs. CONCLUSION: The effect of P. amarus-generated TLY on the immune effector mechanisms of DCs verified its potential to induce an in vitro anti-tumor immune response against the recognized tumor antigen.

Evaluation of Petrifilm Lactic Acid Bacteria Plates for Counting Lactic Acid Bacteria in Food.

Although lactic acid bacteria (LAB) are used widely as starter cultures in the production of fermented foods, they are also responsible for food decay and deterioration. The undesirable growth of LAB in food causes spoilage, discoloration, and slime formation. Because of these adverse effects, food companies test for the presence of LAB in production areas and processed foods and consistently monitor the behavior of these bacteria. The 3M Petrifilm LAB Count Plates have recently been launched as a time-saving and simple-to-use plate designed for detecting and quantifying LAB. This study compares the abilities of Petrifilm LAB Count Plates and the de Man Rogosa Sharpe (MRS) agar medium to determine the LAB count in a variety of foods and swab samples collected from a food production area. Bacterial strains isolated from Petrifilm LAB Count Plates were identified by 16S rDNA sequence analysis to confirm the specificity of these plates for LAB. The results showed no significant difference in bacterial counts measured by using Petrifilm LAB Count Plates and MRS medium. Furthermore, all colonies growing on Petrifilm LAB Count Plates were confirmed to be LAB, while yeast colonies also formed in MRS medium. Petrifilm LAB Count Plates eliminated the plate preparation and plate inoculation steps, and the cultures could be started as soon as a diluted food sample was available. Food companies are required to establish quality controls and perform tests to check the quality of food products; the use of Petrifilm LAB Count Plates can simplify this testing process for food companies.

Naturally occurring immunomodulators with antitumor activity: An insight on their mechanisms of action.

Natural products with immunomodulatory activity are widely used in treatment of many diseases including autoimmune diseases, inflammatory disorders in addition to cancer. They gained a great interest in the last decades as therapeutic agents since they provide inexpensive and less toxic products than the synthetic chemotherapeutic agents. Immunomodulators are the agents that have the ability to boost or suppress the host defense response that can be used as a prophylaxis as well as in combination with other therapeutic modalities. The anticancer activity of these immunomodulators is due to their anti-inflammatory, antioxidant, and induction of apoptosis, anti-angiogenesis, and anti-metastasis effect. These natural immunomodulators such as genistein, curcumin, and resveratrol can be used as prophylaxis against the initiation of cancer besides the inhibition of tumor growth and proliferation. Whereas, immunostimulants can elicit and activate humoral and cell-mediated immune responses against the tumor that facilitate the recognition and destruction of the already existing tumor. This review represents the recent studies on various natural immunomodulators with antitumor effects. We have focused on the relationship between their anticancer activity and immunomodulatory mechanisms. The mechanisms of action of various immunomodulators such as polyphenolic compounds, flavonoids, organosulfur compounds, capsaicin, vinca alkaloids, bromelain, betulinic acid and zerumbone, the affected cancerous cell lines in addition to the targeted molecules and transcriptional pathways have been review and critically analyzed.