Influence of oxygen availability on expression of glutaminolysis genes in human colon cancer cells

Introduction Glutaminolysis, beside glycolysis, is a key metabolic pathway of a cancer cell that provides energy and substrates for the synthesis of nucleic acids, proteins, and lipids. The pathway is mediated by both mitochondrial and cytosolic enzymes. Neither expression of glutaminolysis enzymes in colon cancer cells nor the influence of various oxygen concentrations on their expression has been studied so far. Objectives The aim of the study was to determine and compare the mRNA expression of enzymes involved in glutaminolysis at various oxygen levels in human primary (SW480) and metastatic (SW620) colon cancer cells cultured in 1% O2 (hypoxia), 10% O2 (tissue normoxia), 21% O2 (atmospheric normoxia). Methods Cell viability was determined by Trypan Blue exclusion (TB) and Thiazolyl Blue Tetrazolium Bromide (MTT). The expression of HIF1α, GLUT1, GLS1, AST1, AST2, ACL, PC and GC1, GC2 at mRNA levelwas determined by RT-qPCR. Results. Correlation between increasing oxygen concentration and cell count was not observed. In both cell lines the number of viable cells was the lowest at 10% oxygen. The enzyme profile and expression of proteins involved in glutaminolysis varied depending on oxygen pressure and type of cell lines. In summary, our findings suggest differences in metabolic adaptation to oxygen availability in vivo between primary and metastatic colon cancer cells.

MiR-138-5p Suppresses Cell Growth and Migration in Melanoma by Targeting Telomerase Reverse Transcriptase

Recent evidence suggests the existence of a miRNA regulatory network involving human telomerase reverse transcriptase gene (hTERT), with miR-138-5p playing a central role in many types of cancers. However, little is known about the regulation of hTERT expression by microRNA (miRNAs) in melanocytic tumors. Here, we investigated the effects of miR-138-5p in hTERT regulation in melanoma cells lines. In vitro studies demonstrated higher miR-138-5p and lower hTERT messenger RNA (mRNA) expression in human epidermal melanocytes, compared with melanoma cell lines (A2058, A375, SK-MEL-28) by quantitative polymerase chain reaction (qPCR) observing a negative correlation between them. A2058 melanoma cells were selected to be transfected with miR-138-5p mimic or inhibitor. Using luciferase assay, hTERT was identified as a direct target of this miRNA. Overexpression of miR-138-5p detected by Western blot revealed a decrease in hTERT protein expression (p = 0.012), and qPCR showed a reduction in telomerase activity (p < 0.001). Moreover, suppressions in cell growth (p = 0.035) and migration abilities (p = 0.015) were observed in A2058-transfected cells using thiazolyl blue tetrazolium bromide and flow cytometry, respectively. This study identifies miR-138-5p as a crucial tumor suppressor miRNA involved in telomerase regulation. Targeting it as a combination therapy with immunotherapy or targeted therapies could be used in advanced melanoma treatment; however, more preclinical studies are necessary.

In vitro cytotoxicity investigations of chloroform extract of Anisomeles malabarica on vero and lung cancer [A-549] cell lines

The main objective of this study is to investigate the cytotoxic potential of chloroform extract of medicinal plant Anisomeles malabarica. Successive solvent extraction of Anisomeles malabarica in chloroform was done. The extracts were tested against normal cell lines (Vero) human lung cancer cell lines (A-549) using the thiazolyl blue tetrazolium bromide test (MTT) assay. The results of the present investigation revealed that the chloroform extract of Anisomeles malabarica shown anti-lung cancer activity. The evaluation of the toxicity of medicinal plants and their herbal preparations is essential to determine the applicability of the samples as pharmacological drugs. Further studies are necessary for more extensive pharmacological and toxicological evaluations.

Multi-drug resistance and biofilm production among diarrheagenic Escherichia coli pathotypes isolated from stools of children with acute diarrheal disease

Background. Diarrheagenic E. coli (DEC) is an etiological agent of childhood diarrhea. Resistance against commonly used drugs in the empirical treatment of enteric infections has increased among DEC. Relationship between antibiotic resistance and biofilm formation in microorganisms have been widely reported. This study was aimed to determine the antibiotic resistance and biofilm production pattern among DEC pathotypes isolated from stools of children aged 0–5 years with acute diarrheal disease in Abakaliki, Nigeria. Materials and methods. Diarrheal stool samples were obtained from 60 children and E. coli were isolated and identified using standard guidelines provided for laboratory diagnosis of enteric pathogens. Molecular identification was done by amplification of E. coli universal stress protein A (uspA) using polymerase chain reaction (PCR) method. Detection of virulent genes of DEC pathotypes was performed in a group of multiplex PCR using their specific primers. Kirby–Bauer disk diffusion method was used to determine the antibiotic susceptibility patterns of the isolates while biofilms production was detected by thiazolyl blue tetrazolium bromide dye in a 96-well plate. Results. DEC was isolated in 40 stools among which EIEC [40% (n = 16)] was commonly detected followed by ETEC [30% (n = 12)], EAEC [20% (n = 8)] and typical EPEC [10% (n = 4)]. Half of EAEC showed the highest multidrug resistance against ampicillin, cefoxitin, ciprofloxacin, levofloxacin, and tetracycline with the strongest biofilm production followed by all the EPEC which were resistant to ampicillin, ciprofloxacin, levofloxacin, and tetracycline with moderate biofilm production. All the LT-ETEC exhibited the least resistance to ampicillin and tetracycline with the weakest biofilm production. Conclusion. High frequency of the EIEC pathotype suggests its role as the primary etiological agent of diarrhea in children. Correlation between high drug resistance and biofilm production among the pathotype may indicate that biofilms may provide compatible uptake of resistance genes.

Chrysin and rutin protect against hydrogen peroxide and tert-butyl hydroperoxide induced oxidative cell damage

Objective: Chrysin and rutin are two dietary flavonoids lying in fruits or honey bee’s products. Their pharmacological properties include antioxidant, anti-inflammatory, anticancer, neuroprotection and immunomodulatory. In the current study, the potentiality of chrysin and rutin to protect human gingival fibroblasts against oxidative cell damage has been investigated in vitro. Method: Human gingival fibroblasts, passage 3, were concomitantly put in contact with the cytotoxic compounds and chrysin or rutin for 24 h at 37 °C, 5% CO2 atmosphere, and 96% humidity. The amount of viable cell after the incubated time was recorded by using the thiazolyl blue tetrazolium bromide (MTT) assay. Results: Chrysin in all tested concentration didn’t exhibit any cytoprotective effect against the tert-butyl hydroperoxide-induced oxidative cell damage. Moreover, chrysin in a low concentration (5 and 10 µg/mL) didn’t protect the fibroblasts against oxidative cell damage induced by the hydrogen peroxide. However, chrysin in a concentration of 20 µg/mL showed a significant cytoprotective activity in the hydrogen peroxide-induced cell damage (p < 0.05). Rutin in all tested concentrations protected fibroblasts against hydrogen peroxide and tert-butyl hydroperoxide-induced oxidative cell damage. The cytoprotective effect of rutin didn’t increase with the increase of the concentration when hydrogen peroxide is used to induce oxidative cell damage. However, rutin has protected cells against the tert-butyl hydroperoxide cytotoxicity in a concentration dependent manner. Conclusion: Given to the interesting cytoprotective activities exhibited by chrysin and rutin, further investigations to highlight their cytoprotective involved mechanisms are justified. Keywords: Chrysin, Cytoprotective, Fibroblasts, Rutin.

Effect of Probenecid on Endothelial Cell Growth Rate and Retinal Angiogenesis in an Oxygen-Induced Retinopathy Model

Objectives: Probenecid is an anion transport inhibitor, which, according to the connectivity map (CMap; a biological application database), interferes with hypoxia-induced gene expression changes in retinal vascular endothelial cells (ECs). Here, we investigated the influence of probenecid on retinal EC cytotoxicity and retinal neovascularization in a murine oxygen-induced retinopathy (OIR) model.Methods: The retinal EC growth rate in the presence of hypoxia-mimicking concentrations of cobalt chloride (CoCl2) was determined using the thiazolyl blue tetrazolium bromide (MTT) assay and proliferating cell nuclear antigen (PCNA) expression. In OIR rats, probenecid was administered by intraperitoneal injection (i.p.) from postnatal day (P) 1 to P7. The concentrations of vitreous humor vascular endothelial growth factor (VEGF), hypoxia-inducible factor (HIF)-1α, and placental growth factor (PlGF) were determined by using the ELISA kit at P21. The amount of newly formed vascular lumen was evaluated by histopathological examination. Retinopathy and neovascularization were assessed by scoring isolectin B4 fluorescein–stained retinal flat mounts. Western blots for liver tissue HIF-1α and hepcidin (HAMP) were performed.Results:In vitro, probenecid led to the recession of the hypoxia-induced EC growth rate. In vivo, compared to the OIR retina, the upregulation of VEGF, HIF-1α, and PlGF in phase II retinopathy of prematurity (ROP) was inhibited by probenecid administration. Moreover, probenecid ameliorated neovascularization and resulted in significantly reduced relative leakage fluorescence signal intensity in fluorescein-stained retinal flat mounts (p &lt; 0.05). Probenecid alleviated the liver overactivation of HAMP and downregulation of HIF-1α in OIR rats.Conclusions: This is the first demonstration that implies that probenecid might be a protective compound against retinal angiogenesis in OIR. These changes are accompanied with decreased hyperoxia-mediated hepcidin overproduction. Although the relevance of the results to ROP needs further research, these findings may help establish potential pharmacological targets based on the CMap database.

Nanemulsions of foodstuffs and their nutritiological and valleological characteristics

Annotation. The use of nanotechnology in the medical, food, pharmaceutical, biotechnology industries today is an important scientific progress and valuable human heritage. Nanoemulsion technology is an ideal method for the manufacture of encapsulating systems for functional compounds, as it prevents their biotechnological biodegradation and improves their functional availability in the cells of the body. The aim of the article is a scientific-theoretical and practical review of the nutritional and valeological properties of nanoemulsions, their use for encapsulation of various nutraceuticals, namely fat-soluble vitamin D. The in vitro experiment was performed using Franz diffusion cells to study the release of bioactive compounds from nanocarriers. The cytotoxicity of nanoemulsions was investigated by analyzing the proliferation of thiazolyl blue tetrazolium bromide (TTB) cells and nasal epithelial cells as an “in vitro” model. The article provides to characterize the nutritional and valeological properties of nanoemulsions and to experimentally investigate hydrogels based on nanoemulsions as biocarriers of vitaminized compounds. During the study, low- and high-energy nanoemulsions were created, which were used for encapsulation of vitamin D3 and biologically active supplement - curcumin. Loaded nanoemulsions are added to homopolymer and copolymer hydrogels based on polysaccharides and their combinations. Both nanoemulsions and hydrogels are structurally characterized to evaluate the effect of the composition on the emulsification process by their properties. The cytotoxic effect of nanoemulsions "in vitro" on the epithelium of nasal cells, which had a positive therapeutic effect, was studied. In the future, further exploration and research will investigate the use of nanoemulsions as biocarriers for other vitamins and bioactive substances.

Therapeutic Mechanism of Lapatinib Combined with Sulforaphane on Gastric Cancer

Background. Lapatinib is a small-molecule tyrosine kinase inhibitor that plays important roles in cell proliferation and survival. Administration of lapatinib with capecitabine is an effective treatment for HER2-positive metastatic BC. However, the effects of lapatinib on gastric cancer (GC) remain to be clear. In this study, we aimed to investigate the therapeutic effects of lapatinib combined with sulforaphane on GC and its underlying mechanisms. Methods. SGC-7901 and lapatinib-resistant SGC-7901 cells were treated with lapatinib (0.2 μM), sulforaphane (5 μM), or their combinations. Cell viability, invasion, cycle, and apoptosis of SGC-7901 and lapatinib-resistant SGC-7901 cells were evaluated by thiazolyl blue tetrazolium bromide (MTT), Boyden chamber assay, and flow cytometer. The protein expressions of HER-2, p-HER-2, AKT, p-AKT, ERK, and p-ERK were detected by Western blotting. Results. We observed that lapatinib combined with sulforaphane significantly decreased cell viability and inhibited cell migration of drug-sensitive and drug-resistant cells. Lapatinib sulforaphane also remarkably induced cell apoptosis with G0/G1 arrest. In addition, Western blotting revealed that the expressions of HER-2, p-HER-2, AKT, p-AKT, ERK, and p-ERK were downregulated by lapatinib-sulforaphane treatment. Conclusion. Combination of lapatinib and sulforaphane might be a novel and promising therapeutic treatment for lapatinib-sensitive or lapatinib-resistant GC patients.

Synthesis, Crystal Structure and Bioactivity of Phenazine-1-carboxylic Acylhydrazone Derivatives

A phenazine-1-carboxylic acid intermediate was synthesized from the reaction of aniline and 2-bromo-3-nitro-benzoic acid. It was then esterified and reacted with hydrazine hydrate to afford phenazine-1-carboxylic hydrazine. Finally, 10 new hydrazone compounds 3a–3j were obtained by the condensation reaction of phenazine-1-carboxylic acid hydrazide and the respective aldehyde-containing compound. The structures were characterized by 1H and 13C NMR spectroscopy, MS and single crystal X-ray diffraction. The antitumor activity of the target compounds in vitro (HeLa and A549) was determined by thiazolyl blue tetrazolium bromide. The results showed that compound (E)-N′-(2-hydroxy-4-(2-(piperidine-1-yl) ethoxy) benzyl) phenazine-1-carbonyl hydrazide 3d exhibited good cytotoxic activity.

Synergistic effect of cold atmospheric pressure plasma and free or liposomal doxorubicin on melanoma cells

The aim of the present study was to investigate combined effects of cold atmospheric plasma (CAP) and the chemotherapeutic drug doxorubicin (DOX) on murine and human melanoma cells, and normal cells. In addition to free drug, the combination of CAP with a liposomal drug (DOX-LIP) was also studied for the first time. Thiazolyl blue tetrazolium bromide (MTT) and Trypan Blue exclusion assays were used to evaluate cell viability; the mechanism of cell death was evaluated by flow cytometry. Combined treatment effects on the clonogenic capability of melanoma cells, was also tested with soft agar colony formation assay. Furthermore the effect of CAP on the cellular uptake of DOX or DOX-LIP was examined. Results showed a strong synergistic effect of CAP and DOX or DOX-LIP on selectively decreasing cell viability of melanoma cells. CAP accelerated the apoptotic effect of DOX (or DOX-LIP) and dramatically reduced the aggressiveness of melanoma cells, as the combination treatment significantly decreased their anchorage independent growth. Moreover, CAP did not result in increased cellular uptake of DOX under the present experimental conditions. In conclusion, CAP facilitates DOX cytotoxic effects on melanoma cells, and affects their metastatic potential by reducing their clonogenicity, as shown for the first time.