Extracts of Scrophularia frigida Boiss display potent antitumor effects in human breast cancer cells by inducing apoptosis and inhibition of expression of the human epidermal growth factor receptor 2.

Some species of the Scrophularia genus have been extensively used as a natural remedy for treatment of various medical conditions. The objective of this study was to evaluate the growth inhibitory activity of Scrophularia frigida Boiss extracts as well as to study the effect of the potent extracts on the induction of apoptosis and cell cycle arrest on human breast cancer cells. S. frigida Boiss extracts exhibited obvious inhibitory effects on the growth of cancer cells and induced apoptosis. It is suggested that the extracts exert their anti-proliferative effect through multiple implications such as suppressing growth, arresting the cell cycle, increased DNA fragmentation, downregulation of the expression of human epidermal growth factor receptor 2 and myeloid cell Leukemia-1, and upregulation of pro-apoptotic messenger RNAs like caspase-3 and caspase-9. Taken together, the results obtained indicate that S. frigida Boiss extracts may contain effective compounds that can be used as a therapeutic anticancer agent.

Urtica dioica dichloromethane extract induce apoptosis from intrinsic pathway on human prostate cancer cells (PC3).

Prostate cancer is considered as the major cause of death among men around the world. There are a number of medicinal plants triggering apoptosis response in cancer cells, thus have a therapeutic potential. Therefore, further studies to characterize beneficial properties of these plants in order to introduce novel anti-cancer drugs are the interest of recent researches on the alternative medicine. On the other hand, due to traditional uses and availability of Urtica dioica extract, we decided to evaluate the efficacy of this medicinal herb on pc3 prostate cancer cell line. In the present study the cytotoxic effects of Urtica dioica extract were assessed by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay and trypan blue viability dye. Then, DNA fragmentation and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay were exploited to measure cell death and apoptosis stage. The expression levels of caspase 3, caspase 9 and Bcl-2 genes were quantified by Real-Time PCR. Finally, Cell cycle was analyzed by flow cytometry. MTT assay showed that dichloromethanolic extract of Urtica dioica significantly inhibited the cell growth. According to the DNA fragmentation and TUNEL assay results, the herbal extract was able to induce apoptosis in prostate cancer cells. Our findings also demonstrated that the plant extract substantially increases the caspase 3 and 9 mRNA expression, while decreases Bcl-2. Cell cycle arrest was occurred in G2 stage, due to the results of flow cytometry. These results indicate that dichloromethanolic extract of Urtica dioica can successfully induce apoptosis in PC3 cells. Therefore, it could be used as a novel therapeutic candidate for prostate tumor treatment.

Effects of Urtica dioica dichloromethane extract on cell apoptosis and related gene expression in human breast cancer cell line (MDA-MB-468).

Breast cancer is the most common cancer among women in worldwide, especially in developing countries. Therefore, a large number of anticancer agents with herbal origins have been reported against this deadly disease. This study is the first to examine the cytotoxic and apoptotic effects of Urtica dioica in MDA-MB-468, human breast adenocarcinoma cells. The 3-(4,5-dimethylethiazol-2 yl)-2,5- diphenyltetrazolium (MTT) reduction and trypan-blue exclusion assay were performed in MDA-MB-468 cells as well as control cell line L929 to analyze the cytotoxic activity of the dichloromethane extract. In addition, Apoptosis induction of Urtica dioica on the MDA-MB-468 cells was assessed using TUNEL (terminal deoxy transferase (TdT)-mediated dUTP nick- end labeling) assay and DNA fragmentation analysis and real-time polymerase chain reaction (PCR). The results showed that the extract significantly inhibited cell growth and viability without inducing damage to normal control cells. Nuclei Staining in TUNEL and DNA fragments in DNA fragmentation assay and increase in the mRNA expression levels of caspase-3, caspase-9, decrease in the bcl2 and no significant change in the caspase-8 mRNA expression level, showed that the induction of apoptosis was the main mechanism of cell death that induce by Urtica dioica extract. Our results suggest that urtica dioica dichloromethane extract may contain potential bioactive compound(s) for the treatment of breast adenocarcinoma.

Micro-RNAs: The new potential biomarkers in cancer diagnosis, prognosis and cancer therapy.

MicroRNAs (miRNAs) are a large class of small noncoding RNAs approximately 22 nucleotides in length. They are the main regulators of gene expression, regulating specific oncogenes, tumor suppressors, cancer stem cells and metastasis. MicroRNAs have become valuable to cancer research in recent years. They appear as a significant biomarker in tumorigenesis. Briefly, the capacities of miRNA to identify between tumor and normal tissue, to distinguish between various subgroups of tumors and to foretell results or responses to therapy have attracted scientist's attention to these small RNAs. MicroRNAs' remarkable stability in both the tissue and bloodstream of cancer patients has elevated the possibility that miRNAs may prove to be a novel diagnostic biomarker. This review focuses on the utility of miRNAs as key biomarkers in cancer diagnosis, cancer prognosis and cancer therapy.

siRNA-mediated silencing of MDR1 reverses the resistance to oxaliplatin in SW480/OxR colon cancer cells.

One of the most challenging aspects of colon cancer therapy is rapid acquisition of multidrug resistant phenotype. The multidrug resistance gene 1 (MDR1) product, p—glycoprotein (P—gp), pump out a variety of anticancer agents from the cell, giving rise to a general drug resistance against chemotherapeutic agents. The aim of this study was to investigate the effect of a specific MDR1 small interference RNA (siRNA) on sensitivity of oxaliplatin—resistant SW480 human colon cancer cell line (SW480/OxR) to the chemotherapeutic drug oxaliplatin. SW480 cells were made resistant by continuous incubation with stepwise serially increased concentrations of oxaliplatin over a 6—months period. Resistance cell were subsequently transfected with specific MDR1 siRNA. Relative MDR1 mRNA expression was measured by Quantitative real—time PCR. Western blot analysis was performed to determine the protein levels of P—gp. The cytotoxic effects of oxaliplatin and MDR1 siRNA, alone and in combination were assessed using MTT and the number of apoptotic cells was determined with the TUNEL assay. MDR1 siRNA effectively reduced MDR1 expression in both mRNA and protein levels. MDR1 down—regulation synergistically increased the cytotoxic effects of oxaliplatin and spontaneous apoptosis SW480/OxR. Our data demonstrates that RNA interference could down regulate MDR1 gene expression and reduce the P—gp level, and partially reverse the drug resistance in SW480/OxR cells in vitro. Therefore, the results could suggest that MDR1 silencing may be a potent adjuvant in human colon chemotherapy.

The effect of canola meal tannins on the intestinal absorption capacity of broilers using a D-xylose test.

In three D-xylose absorption experiments, the effect of 1% HCl/methanol, 70% methanol or 70% acetone extracts of canola meal (CM) or 70% acetone extract of soybean meal (SBM) containing polyphenols, phenolic acids, tannins and phytic acid on intestinal absorption capacity of broilers was determined. In Exp. 1, the experimental groups received orally D-xylose solution alone or with methanol/HCl, methanol or acetone extracts of CM. In Exp. 2, the experimental groups received D-xylose alone or with acetone extracts of CM or SBM. In Exp. 3, the experimental groups received D-xylose plus sucrose solution or D-xylose plus acetone extracts of CM or SBM. In Exps. 2 and 3, the CM extracts contained 2.7 and 2.6, 2.4 and 2.3, 3.2 and 3.2, and 2.4 and 2.2 times higher polyphenols, phenolic acids, tannins and condensed tannins than the corresponding SBM extracts respectively. Blood samples were collected in 40-min intervals, and plasma D-xylose was measured. Compared to the Control, plasma D-xylose in Exp. 1 was lower (p < 0.001) by 81, 69 and 73% at 40-min, by 41, 44 and 37% at 80-min and by 22, 31, and 23% at 120-min post-ingestion of the HCl/methanol, methanol and acetone extracts respectively. In both Exps. 2 and 3, plasma D-xylose level was lower (p < 0.001) in groups dosed with CM extract or SBM extract at each time of blood collection, when compared to the respective Control group. However, in Exp. 3, birds dosed with SBM extract had higher plasma D-xylose than CM extract-dosed birds by 28, 8 and 21% at 40, 80 and 120 min respectively (p < 0.01). In conclusion, although CM extract caused a lower absorption of D-xylose, based on 5 to 10% of CM inclusion levels in practical broiler rations, the soluble bioactive components of CM will likely have minor impact on the absorption capacity of the chicken intestine.

Evaluating the resistance of eimeria spp. Field isolates to anticoccidial drugs using three different indices.

BACKGROUND: In this study, the presence of resistance to diclazuril, amprolium+ethopabate and salinomycin, representing some of the commonest anticoccidials in Iran's poultry industry, against three mixed Eimeria field isolates were investigated. METHODS: Three Eimeria field isolates, collected from typical broiler farms in Iran, were propagated once, inoculated to 480 broilers, comprising 30 chicks in each treatment. The non-medicated or medicated diets containing one of the above mentioned anticoccidials were provided ad-lib. Drug efficacy was determined using the Global index (GI), Anticoccidial Sensitivity Test (AST) and Optimum Anticoccidial Activity (OAA). RESULTS: None of the field isolates were fully sensitive to the selected anticoccidials. All isolates showed reduced sensitivity/partial resistance to salinomycin. Resistance to amprolium+ethopabate was evident and partial to complete resistance was recorded for diclazuril. CONCLUSION: Limited efficacy of the selected anticoccidials is obvious. Considering the cost of continuous use of anticoccidials in the field, altering the prevention strategy and rotation of the anticoccidials with better efficacy, would prevent further economic losses induced by coccidiosis.

The effect of tannic acid and polyethylene glycol on the absorption capacity of chicken intestine for d-xylose and ß-carotene.

In an experiment, the possible influence of tannic acid (TA) and polyethylene glycol (PEG) on the absorption capacity of intestine for d-xylose and ß-carotene in broiler chicken was investigated. Four groups of nine 28-day-old broiler cockerels received d-xylose (500 mg) and ß-carotene (52 µg) solutions (Group 1 to 4) with TA (1 g, Group 2 to 4) and PEG (500 mg Group 3 and 1 g Group 4), orally. One blood sample prior to, and four others after the administration of test materials, were collected from wing vein on 40 min basis, for 160 min and the concentration of plasma d-xylose was determined. The concentration of ß-carotene was also measured in plasma of blood samples taken prior to and 160 min post-administration of the test materials. Plasma d-xylose concentration of all groups showed quadratic correlations with time (p < 0.001, r(2) = 0.84, 0.60, 0.70 and 0.74 for Group 1 to 4, respectively). Administration of TA reduced the plasma concentration of d-xylose in Group 2. However, feeding PEG after TA raised the concentration of d-xylose in Group 4 to the level that there was no difference in that variable between this group and Group 1. Although the plasma concentration of ß-carotene was increased in 160 min post-ingestion of the test material, no difference was found in that variable among the experimental groups. In conclusion, TA and its interaction with PEG have impacts on the absorption capacity of intestine for d-xylose and highly likely other simple sugars, but TA or PEG have no influence on the absorption of ß-carotene and most probably other fat soluble vitamins.

Biopathologic Characterization of Three Mixed Poultry Eimeria spp. Isolates.

BACKGROUND: Coccidiosis of domestic fowl, caused by species of the Genus Eimeria, is responsible for important economic losses in poultry production. Because different species and/or strains can vary in pathogenicity and other biological parameters, their precise characterization is important for epizootiological studies. METHODS: Fifty samples from litter, whole intestinal tract and feces were collected from poultry houses located in different provinces of Iran. One hundred twenty male day-old broiler chicks were challenged with three selected isolates. Data on weight gain, Food Conversion Ratio (FCR), food intake, lesion scoring and shedding of oocysts per gram of feces were recorded and analyzed by the Duncan's test. RESULTS: In all treatments, the challenged groups had statistically significant lower weight gain than that of unchallenged control group. Isolate three caused the lowest weight gain and food intake and the worst lesion score as well as FCR. Despite originating from close geographical regions for isolates 1 and 2, the difference in biopathologic factors may be either due to different proportion of identified species or the different pathogenicity of the species present in the isolates. CONCLUSION: The results highlight the importance of considering various species of Eimeria in designing the preventive, control and treatment strategies to prevent coccidiosis in different regions of Iran. Further characterization of each isolate would be the next step to provide a basis for coccidiosis research with well-characterized local isolates.

D-Xylose absorption capacity of broiler intestine in response to phytic acid.

1. Dietary phytic acid (PA) reduces the apparent digestion and of dietary nutrients, increases the excretion of endogenous amino acids and minerals and reduces the concentration of blood glucose. 2. An experiment was conducted to examine the effect of phytic acid on the absorption capacity of the intestine in broiler chicken, using a D-xylose absorption test. 3. Three groups of ten 26-d-old apparently healthy broiler cockerels (Ross 308) were dosed with D-xylose solution (500 mg/kg BW, Group 1) or D-xylose solution + PA (330 or 660 mg/5 ml/bird, in groups 2 and 3), respectively. The plasma concentration of D-xylose was measured at 40-min intervals after ingestion of test materials, for a total of 160 min. 4. There was a quadratic correlation between the concentration of plasma D-xylose and time in all experimental groups (P < 0001, R(2)= 078, 080 and 081 for groups 1-3, respectively). Ingestion of PA at 660 mg reduced the concentration of plasma D-xylose by 216 and 105% at 40 and 80 min after ingestion of the test material, indicating a lower absorption of this sugar. 5. It was concluded that dietary phytates might affect the productive performance of chicken, at least partly, by disturbing the transport mechanisms involved in the absorption of nutrients.

Influence of tannic acid and polyethylene glycol on the excretion and digestibility of amino acids in gelatin-fed broilers.

1. A precision feeding experiment was conducted to evaluate the effects of tannic acid (TA) and polyethylene glycol (PEG) on the excretion of amino acids, and the apparent and true digestibility of gelatin protein in broilers. 2. In a 3 x 2 x 2 factorial design, ninety-six 7-week-old broiler cockerels in 8 replicates of 12 treatments, were fed on warm solution of gelatin alone (0, 6 or 12 g/50 ml/bird, Treatments 1-3), or gelatin with TA solution (4.5 g TA/10 ml/bird, Treatments 4-6) and PEG solution (2 g/10 ml/bird, Treatments 7-12). Total excreta were collected for 48 h and the amino acid contents of gelatin and excreta were determined. 3. In the absence of TA, the digestibility of gelatin was almost complete. TA increased the excretion of amino acids from gelatin-fed birds to varying extents. Although the digestibility of all indispensable and dispensable amino acids was adversely affected by the presence of TA, increasing the amount of gelatin from 6 to 12 g improved the apparent and true digestibility of amino acids. PEG reduced the excretion of amino acids and improved the digestibility of amino acids in gelatin in TA-dosed birds. However, the effect was greater when the lesser amount of gelatin was fed. 4. In conclusion, PEG seemed to play an important role in reducing the effect of dietary tannins in the gastrointestinal tract of birds fed on diets high in tannins and improved protein digestibility and utilisation, particularly when the diet was low or marginal in protein.

Influence of dietary tannic acid and polyethylene glycol on growth and intestinal D-xylose absorption of broiler cockerels and activity of serum enzymes.

1. In an experiment on broiler cockerels, the influence of tannic acid (TA) and polyethylene glycol (PEG) on body weight gain (BWG), feed conversion ratio (FCR), weight of intestine and liver, the activities of serum enzymes LDH, AST, ALT and intestinal absorption function were investigated. 2. Broiler cockerels were given either a commercial diet alone (control group) or a commercial diet with TA (20 g/kg), PEG (10 g/kg) or TA plus PEG (20 + 10 g/kg), for 10 d. 3. On the last day of the experiment, all birds and remaining feed were weighed individually and a sample of blood was taken to measure the serum activities of lactate dehydrogenase (LDH), aspartate aminotransferase (AST) and alanine aminotransferase (ALT). The capacity of intestinal cells for the absorption of D-xylose was measured. Finally all birds were killed humanely and the intestine and liver were weighed. 4. The results showed that TA significantly reduced BWG and FCR, as well as the activity of LDH, AST and ALT. 5. TA also increased the relative weight of the intestine. Adding PEG alone had no effect on any of the measured parameters. 6. However, PEG improved significantly BWG, FCR and the activity of LDH and AST of TA-fed birds. 7. The plasma D-xylose concentration of experimental birds was similar for all dietary treatments most likely because of temporal separation between feeding the dietary TA and administering the D-xylose. 8. It was concluded that the presence of tannins in the GI lumen of the bird was necessary to affect the processes involved in the absorption of simple sugars such as D-xylose, at the level of intestinal absorptive cells.

Hydropathic influences on the quantification of equine heart cytochrome c using relative ion abundance measurements by electrospray ionization fourier transform ion cyclotron resonance mass spectrometry.

The number of publications documenting the utility of electrospray ionization (ESI) Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) for the analysis of biological molecules has increased in geometric proportion spanning diverse areas of research. Currently, we are investigating the capabilities of ESI-FTICR to quantify relative molecular ion abundances of biopolymers, an area which has not been explored rigorously. We present here the results of an investigation of a two-component system utilizing equine heart cytochrome c (EH) as the analyte and bovine heart cytochrome c (BH) as a constant concentration internal standard. As these compounds are relatively large ( approximately 12 kDa), they will become multiply charged during the electrospray process. Using appropriate solution and instrument conditions, the 7(+) and 8(+) charge states were enhanced for both cytochrome c species. We report that using the average of the ion abundances for the two charge states observed for each species, the linear curve (intensity ratio vs concentration ratio) had a dynamic range of 0.045-2.348 microM (1.7 orders of magnitude). Linear least-squares regression analysis (LLSRA) of these averaged ion abundances (i.e. [(EH + 7H(+))(7+)/(BH + 7H(+))(7+) + (EH + 8H(+))(8+)/(BH + 8H(+))(8+)]/2) yielded the equation y = 1.005x + 0.027. The slope of the line with its calculated precision, reported as one standard deviation, is 1.005 +/- 0.0150, which is statistically ideal (i.e. equal to unity). However, LLSRA of the ion abundances of the two individual charge states were significantly different (i.e. the slope of the (EH + 7H(+))(7+)/(BH + 7H(+))(7+) peak intensity ratio vs molar ratio data was 0.885 +/- 0.0183 and the slope of the (EH + 8H(+))(8+)/(BH + 8H(+))(8+) data was 1.125 +/- 0.0308). We attribute this difference to the variation in primary amino acid sequence for the two cytochrome c species. Both have 104 amino acids, but there are three residue substitutions between EH and BH; one of the substitutions confers an additional basic site to EH. While this extra basic residue may imply an additional charging site, the low charge states observed under the solution conditions employed indicate that most (>66%) basic sites are not protonated. However, the extra basic site also renders EH slightly more hydrophilic. These results present significant considerations when choosing internal standards for the quantification of large proteins by ESI-FTICR-MS and demonstrate that relative molecular ion signals in FTICR can be used to quantify macromolecular species in the nanomolar regime.

Study of 4-quinolone antibiotics in biological samples by short-column liquid chromatography coupled with electrospray ionization tandem mass spectrometry.

Simultaneous detection and confirmation of 15 quinolone antibiotics was accomplished by fast short-column liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC/MS/MS). Several physiochemical parameters such as hydrophobicity and aqueous dissociation constants were calculated from the structural formulas of the quinolone drugs, and their impact on both chromatographic and mass spectrometric behavior was studied. Additionally, a possible influence of bulk solution pH on electrospray detection sensitivity of 4-quinolones was investigated and compared to predictions based on solution-phase equilibria. A signal intensity comparison of the MH+ ions at different pH values for all 15 compounds did not reveal any pH effect, despite variations by several orders of magnitude in equilibrium concentrations in bulk solution. To demonstrate the potential of the LC/MS/MS method, its application to trace analysis in several biological matrices such as milk, salmon, and human urine was investigated. The method was shown to be sensitive with detection limits down to 1 ppb in both milk and salmon tissue. The versatility of the method was also exhibited by utilizing it for rapid identification of urinary metabolites of ciprofloxacin. Finally, a new complementary approach is described for confirmatory analyses of 4-quinolones by means of a quasi-MS/MS/MS technique involving in-source collision-induced dissociation. It is shown that LC/quasi-MS/MS/MS can significantly enhance structural information and, thus, the specificity of analysis for the investigated 4-quinolones.