Treatment of bleomycin-induced pulmonary fibrosis by inhaled tacrolimus-loaded chitosan-coated poly(lactic-co-glycolic acid) nanoparticles

Pulmonary fibrosis is a chronic lung disease characterized by inflammation and collagen deposition, with an estimated mortality rate exceeding 70%. Here, we evaluated the therapeutic effectiveness of inhaled tacrolimus-loaded chitosan-coated poly(lactic-co-glycolic acid) nanoparticles (chitosan TAC PLGA-NPs) in a bleomycin-induced pulmonary fibrosis mouse model. Chitosan TAC PLGA-NPs were fabricated using an o/w emulsification diffusion method, and uncoated TAC PLGA-NPs and chitosan TAC PLGA-NPs were spherical with approximate diameters of 320 and 441 nm, respectively. The zeta potential of chitosan TAC PLGA-NPs (+13.6 mV) was increased significantly by chitosan-coating versus uncoated TAC PLGA-NPs (−28.3 mV). The incorporation efficiency of tacrolimus was 37.7%, and the tacrolimus was gradually released until about 5 day. Direct inhalation of chitosan TAC PLGA-NPs (TAC 180 μg/mouse) twice a week produced marked anti-fibrotic efficacy in mice with bleomycin-induced pulmonary fibrosis, which was much better than the efficacy resulting from daily oral administration (TAC 300 μg/mouse) on the basis of hematoxylin/eosin and Masson’s trichrome staining assessments. Imaging of lung deposition showed that chitosan TAC PLGA-NPs were located well in the lungs and gradually faded over 96 h. The pulmonary delivery of tacrolimus could be therapeutically efficacious for treating pulmonary fibrosis. TAC-loaded PLGA nanoparticles should be considered to be an efficient sustained-release type inhalation system that reduces administration frequency and relevant side effects.     

Improved cisplatin delivery in cervical cancer cells by utilizing folate-grafted non-aggregated gelatin nanoparticles

PurposeCisplatin is highly effective in the treatment of cervical cancer. However, in therapeutic doses, cisplatin induces several adverse effects due to undesirable tissue distribution. Therefore, it is worth targeting cisplatin in cervical cancer cells by implicating non-aggregated ligand-modified nanotherapeutics.Methods and resultsHere, we report the preparation of non-aggregated folic acid-conjugated gelatin nanoparticles of cisplatin (Cis-GNs-FA) by two-step desolvation method with mean particle size of 210.6 ± 9.6 nm and 140.5 ± 10.9 nm for Cis-GNs to improve the drug delivery in cervical cancer, HeLa cells. FTIR and DSC spectra confirmed the presence and stability of cisplatin in gelatin matrix. Furthermore, amorphization of cisplatin in nanoparticles was ascertained by PXRD. Drug release followed a first-order release kinetic at both pH ∼ 5.6 (cervical cancer pH) and pH ∼ 7.4. In addition, a significant (P < 0.05) decrease in IC₅₀ value (8.3 μM) and enhanced apoptosis were observed in HeLa cells treated with Cis-GNs-FA as compared to Cis-GNs (15.1 μM) and cisplatin solution (40.2 μM). In contrast, A549 lung cancer cells did not discriminate between Cis-GNs-FA and Cis-GNs due to the absence of folate receptors-α (FR-α). Consistently, higher cellular uptake, 80.54 ± 7.60% was promoted by Cis-GNs-FA significantly (two-way ANOVA, P < 0.05) greater than 51.68 ± 9.78%, by Cis-GNs. This was also illustrated by CLSM images, which indicated that Cis-GNs-FA preferably accumulated in the cytoplasm of HeLa cells nearby nucleus by following receptor-mediated endocytosis pathway as compared to Cis-GNs.ConclusionTherefore, Cis-GNs-FA warrants further in-depth in vitro and in vivo investigations to scale up the technology for clinical translation.     

Biological activities of skin and parotoid gland secretions of bufonid toads (Bufo bufo,Bufo verrucosissimus and Bufotes variabilis) from Turkey

Toad glandular secretions and skin extractions contain numerous natural agents which may provide unique resources for novel drug development. Especially the skin-parotoid gland secretions of toads from genus Bufo contain as many as 86 different types of active compounds, each with the potential of becoming a potent drug. In the present study, crude skin-parotoid gland secretions from Bufo bufo, Bufo verrucosissimus and Bufotes variabilis from Turkey were screened against various cancer cells together with normal cells using MTT assay. Furthermore, the antimicrobial properties of skin secretions were tested on selected bacterial and fungal species for assessing the possible medical applications. Antimicrobial activity of skin secretions was studied by determining minimal inhibitory concentration (MIC) in broth dilution method. Hemolytic activity of each skin-secretion was also estimated for evaluating pharmaceutical potential. Both skin-parotoid gland secretions showed high cytotoxic effect on all cancerous and non-cancerous cell lines with IC₅₀ values varying between <0.1 μg/ml and 6.02 μg/ml. MIC results of antimicrobial activity tests were found to be between 3.9 μg/ml and 250 μg/ml. No hemolytic activities on rabbit red blood cells at concentrations between 0.5 μg/ml and 50 μg/ml were observed. In conclusion, skin-parotoid secretions of bufonid toads might be remarkable candidates for anti-cancer and antimicrobial agents without hemolytic activities.     

Automated cerebrospinal fluid cell count — New reference ranges and evaluation of its clinical use in central nervous system infections

ObjectivesThe purposes of this study were to establish new reference ranges for leukocytes in the CSF and to examine if the separation of mononuclear cells into lymphocytes and monocytes could be used to differentiate between various CNS infections that present with a similar picture in manual CSF cell counts.Design and methodsThe automated cell counter Siemens ADVIA 2120i was used. For the reference range section, we analyzed CSF from 80 neurologically healthy volunteers. For the differential diagnosis section we analyzed cell counts and hospital records from 175 patients with CSF mononuclear pleocytosis.ResultsCorrelation was good between automated and manual leukocyte counts for samples with erythrocyte counts < 250 cells/μL. For the neurologically healthy volunteers studied in the reference range section, the 95th percentile was 3.0 cells/μL for lymphocytes, 1.0 cell/μL for monocytes and 1.0 cell/μL for granulocytes. In the differential diagnosis section, comparisons were done between the groups Lyme neuroborreliosis and viral CNS infection. There were no significant differences between these two groups regarding cell counts; neither for lymphocytes, median 58 cells/μL vs. 72 cells/μL (P = n.s.); nor for monocytes, median 13 cells/μL vs. 16 cells/μL (P = n.s.); nor for granulocytes, median 1 cell/μL vs. 2 cells/μL (P = n.s.)ConclusionsWe suggest new CSF cell count reference ranges of < 4 cells/μL for lymphocytes, < 3 cells/μL for monocytes and < 3 cells/μL for granulocytes. The separation of mononuclear cells into lymphocytes and monocytes did not facilitate the discrimination between Lyme neuroborreliosis and viral CNS infection.     

Vitamin E (α-tocopherol) enhances the PDT action of hematoporphyrin derivatives on cervical cancer cells

ObjectiveThe effect of antioxidant vitamin E (VE) on PDT (a mainly reactive oxygen species-driven process) has in the past shown contradicting results. Hence, we studied the effect of different concentrations and different incubation periods of VE on the PDT cytotoxicity of the cervical adenocarcinoma HeLa cell line.Materials and methodsHeLa cells were incubated with 25 μg/ml of hematoporphyrin derivatives (HpD) for 25 min (1st PDT regimen) or 24 h (2nd PDT regimen), then irradiated with visible light (total light dose of 10 J/cm²) either with or without different concentrations of VE (1–1000 μM) which had been incubated with the cells for 1 h or 24 h prior to PDT. After irradiation, viability was measured using MTT assay.ResultsThe results obtained showed that PDT is effective against cervical cancer cells. Incubation of HpD for 24 h leads to improved PDT action. Higher concentrations of VE incubated in HeLa cells for 1 h before the 2nd PDT regimen significantly enhanced cytotoxicity of PDT and the maximal enhancement was at 1000 μM of VE. The cytotoxic effect of VE on HeLa cells after incubation for 24 h before PDT is enhanced by the PDT action.ConclusionIn conclusion 1000 μM of VE can be used 1 h before PDT to enhance its effect on cervical adenocarcinoma, a disease which is steadily increasing in young women. It is well-known that the cervical adenocarcinoma is resistant to anticancer agents and radiotherapy and it was previously considered to be HpD-PDT resistant.     

Hypericin- and mTHPC-mediated photodynamic therapy for the treatment of cariogenic bacteria

ObjectiveDental caries is one of the most common diseases in Western countries. Its pathoetiology is multifactorial, however, bacteria including Streptococcus mutans and the closely related Streptococcus sobrinus are regarded as key factors involved in this process. The fact that therapeutic approaches to eradicate these microorganisms are still limited prompted us to investigate the treatment potential of photodynamic therapy with the photoactive compounds hypericin (HYP) and meso-tetra(hydroxyphenyl)chlorin (mTHPC) in vitro.Material and methodsS. mutans and S. sobrinus were cultivated under standard conditions and incubated with HYP (Invitrogen, Basel, Switzerland), the liposomal mTHPC derivative Foslipos (FOS, Biolitec, Jena, Germany), or a mixture of both at concentrations ranging between 0.625 and 10 μg/ml for various time points. Following a thorough washing step, bacteria were irradiated with a dental polymerization instrument (400–505 nm). All samples were subjected to serial dilutions and spiral plating on blood agar plates. Viable colony counts were determined after 48 h in culture. Photosensitizer fluorescence of bacteria was visualized by confocal microscopic techniques.ResultsOne hundred percent of S. sobrinus could be killed by a 15 min incubation with as little as 2.5 μg/ml HYP, 5 μg/ml FOS or a mixture of 1.25 μg/ml of each photosensitizer followed by light activation of 120 s. In contrast to S. sobrinus, S. mutans displayed a significant dark toxicity for FOS (10–1.25 μg/ml) and no relevant PDT effects using HYP (10–0.625 μg/ml) under these conditions. HYP-mediated PDT effects (10 μg/ml) could be enhanced to more than 99.9% by prolonging photosensitizer incubation to 30 min and fractional illumination (2×120 s). Complete eradication of S. mutans was achieved by incubation for 15 min with a mixture of 0.625 μg/ml each of FOS and HYP and illumination for 120 s.ConclusionFor both S. mutans and S. sobrinus, short PDT protocols with FOS and/or HYP could be established that completely eradicated these cariogenic bacteria in suspension. Our study, however, indicated that careful optimization of PDT conditions may be necessary for successful treatment of even closely related bacterial species. In multispecies microbial populations, the application of photosensitizer combinations for PDT may be useful.     

Diagnostic and prognostic potentials of microRNA-27a in osteosarcoma

AimGrowing evidence suggest that the microRNA (miR)-23a/24-2/27a cluster may play a crucial role in mammary tumorigenesis and act as a novel class of oncogenes. Among these members, miR-27a has been reported to promote proliferation, migration and invasion in human osteosarcoma cells. The aim of this study was to detect the serum levels of miR-27a in osteosarcoma patients and to investigate its associations with clinicopathological features and prognosis.MethodsmiR-27a levels in sera from 166 osteosarcoma patients and 60 healthy controls were detected by real-time quantitative RT-PCR. Then, the associations of serum miR-27a level with clinicopathological factors or survival of osteosarcoma patients were further evaluated.ResultsCompared to healthy controls, the serum levels of miR-27a were significantly increased in osteosarcoma patients (P < 0.001). Importantly, miR-27a could efficiently screen osteosarcoma patients from healthy controls (Area under receiver operating characteristic curve, AUC = 0.867). Then, high miR-27a expression was more frequently occurred in osteosarcoma patients with advanced clinical stage (P = 0.001), positive distant metastasis (P = 0.01) and poor response to chemotherapy (P = 0.008). In Kaplan–Meier survival analysis, high miR-27a expression was a significant indicator for poor overall survival (P = 0.006) as well as poor disease-free survival (P = 0.01). Furthermore, multivariate analysis demonstrated that miR-27a expression was an independent and significant prognostic factor to predict overall survival (P = 0.01) and disease-free survival (P = 0.03).ConclusionmiR-27a expression may be elevated in sera of osteosarcoma patients and in turn contributes to aggressive progression of this malignancy. Detection of serum miR-27a levels may have clinical potentials as a non-invasive diagnostic/prognostic biomarker for osteosarcoma patients.     

Ursolic acid regulates aging process through enhancing of metabolic sensor proteins level

We previously reported that Ursolic Acid (UA) ameliorates skeletal muscle performance through satellite cells proliferation and cellular energy status. In studying the potential role of the hypothalamus in aging, we developed a strategy to pursue UA effects on the hypothalamus anti-aging proteins such as; SIRT1, SIRT6, PGC-1β and α-Klotho.In this study, we used a model of aging animals (C57BL/6). UA dissolved in Corn oil (20 mg/ml) and then administrated (200 mg/Kg i.p injection) to mice, twice daily for 7 days. After treatment times, the mice perfused and the hypothalamus isolated for preparing of tissue to Immunofluorescence microscopy.The data illustrated that UA significantly increased SIRT1 (∼3.5 ± 0.3 folds) and SIRT-6 (∼1.5 ± 0.2 folds) proteins overexpression (P< 0.001). In addition, our results showed that UA enhanced α-Klotho (∼3.3 ± 0.3) and PGC-1β (∼2.6 ± 0.2 folds) proteins levels (P < 0. 01). In this study, data were analyzed using SPSS 16 (ANOVA test).To the best of our knowledge, it seems that UA through enhancing of anti-aging biomarkers (SIRT1 and SIRT6) and PGC-1β in hypothalamus regulates aging-process and attenuates mitochondrial-related diseases. In regard to the key role of α-Klotho in aging, our data indicate that UA may be on the horizon to forestall diseases of aging.     

Molecularly targeted gemcitabine-loaded nanoparticulate system towards the treatment of EGFR overexpressing lung cancer

Molecularly targeted therapy emerged as a novel therapeutic strategy in the treatment of multiple cancers. In the present study, we have developed gemcitabine (GEM)-loaded cetuximab (CET) surface modified poly(lactic) acid (PLA) nanoparticles (NP) (CET-GEM/PLA NP) to target to epidermal growth factor receptor (EGFR) overexpressing non-small cell lung cancer (A549) cells. The resultant CET-GEM/PLA NP showed a very uniform particle size of ∼ 120 nm and spherical morphology. It exhibited a pH-dependent controlled release pattern. A sustained release of drug in the physiological conditions and faster release in tumor pH will greatly improve the chemotherapeutic efficiency of therapeutic system. Higher or enhanced cellular uptake of CET-GEM/PLA NP in A549 cancer cells clearly indicates the EGFR-mediated receptor based active targeting. Nearly, a two-fold increase in fluorescent intensity was observed for CET-GEM/PLA NP comparing to that of non-targeted NP in the cancer cells. EGFR-mediated internalization of the targeted NP was further confirmed by the confocal microscopy. MTT assay clearly showed the enhanced cell killing effect of CET-conjugated NP due to the selective delivery of GEM to the EGFR over expressing cancer cells. Finally, comparing to the non-targeted NP, CET-GEM/PLA NP showed greater level of cell apoptosis (early and late apoptosis ∼ 40%). Our results showed that antibody conjugation on the surface of NP could be a potential treatment strategy for EGFR over expressing cancer cells. This suggests that CET-GEM/PLA NP could be potentially used for the treatment of NSCLC (lung cancers).     

Serum adipocyte fatty acid binding proteins and adiponectin in patients with coronary artery disease: The significance of A-FABP/adiponectin ratio

BackgroundAdipocyte fatty acid binding protein (A-FABP) and adiponectin have been shown to play important roles in atherosclerosis. We investigated serum A-FABP, adiponectin and A-FABP/adiponectin ratio in patients with coronary artery disease (CAD).MethodsA total of 340 subjects who underwent coronary angiography (CAG) were classified into CAD group (n = 211) and non-CAD group (n = 129) according to the CAG. Serum A-FABP and adiponectin concentrations were determined by enzyme-linked immunosorbent assays.ResultsCAD patients tend to have higher A-FABP concentrations than non-CAD subjects, the difference is significant only between female CAD patients and controls [22.8 (18.6–25.7) ng/ml vs 18.1 (15.6–21.8) ng/ml, P = 0.008]. Serum A-FABP concentration was independently associated with Gensini scores in female subjects (P = 0.018). CAD patients have significant higher serum A-FABP/adiponectin ratio [1.51 ± 0.05 vs 0.89 ± 0.03 ng/μg, P < 0.01] than controls in both genders.ConclusionsSerum A-FABP is associated with CAD more closely in female than in male. The A-FABP/adiponectin ratio may be a more useful indicator for CAD than A-FABP or adiponectin alone.     

Evaluation of anti-diabetic and anti-tumoral activities of bioactive compounds from Phoenix dactylifera L’s leaf: In vitro and in vivo approach

Among various chronic disorders, cancer and diabetes mellitus are the most common disorders. This study was designed to evaluate the effectiveness of hydroalcoholic extract of Phoenix dactylifera L. leaves (HEPdL) in animal models of type II diabetes in vitro/in vivo and in a human melanoma-derived cell line (IGR-39). A liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis was also performed to determine the amount of phenolic and flavonoid compounds in this plant. The physicochemical results by LC–MS/MS analysis of HEPdL showed the presence of 10 phenolic compounds. The in vitro study showed that the extract exhibited a more specific and potent inhibitor of α-glucosidase than α-amylase with an IC₅₀ value of 20 ± 1 μg/mL and 30 ± 0.8 μg/mL, respectively. More importantly, the in vivo study of the postprandial hyperglycemia activity with (20 mg/kg) of HEPdL showed a decrease in plasma glucose levels after 60 min in resemblance to the glucor (acarbose) (50 mg/kg) effect. The oral administration of HEPdL (20 mg/kg) in alloxan-induced diabetic mices for 28 days showed a more significant anti-diabetic activity than that of the drug (50 mg/kg). Moreover, cytotoxicity effects of HEPdL in IGR-39 cancer cell lines were tested by MTT assay. This extract was effective in inhibiting cancer cells growth (IGR-39) at dose 35 and 75 μg/mL. These results confirm ethnopharmacological significance of the plant and could be taken further for the development of an effective pharmaceutical drug against diabetes and cancer.     

Directional modification of chrysin for exerting apoptosis and enhancing significantly anti-cancer effects of 10-hydroxy camptothecin

Chrysin, one of natural flavonoid compounds, has recently been found to possess anti-inflammatory, antiallergic and anticancer properties. To increase its anticancer effects, 5 chrysin derivates were synthesized on the base of DNA intercalator structure. The inhibiting effects of chrysin and its derivatives on cancer cells Hela, BGC823, MCF-7, HepG2, and normal cells HEK-293, were evaluated by MTT assays. 5-(2′-amino) phenyl-7-cyclohexanemethylchrysin (Ch-1), a unique chrysin derivate, killed all the cancer cells but kept above 60% survival rate in normal cells HEK-293 at 62.5 μM. Treated with chrysin from 250 μM to 500 μM, those cells were still maintained above 60% survival rate. The result of circular dichroism spectra showed that Ch-1 could intercalate DNA while chrysin had no effects on DNA. Interestingly, Hela cells survival rates were 95% and 10%, after treated with 20 μM and 30 μM of Ch-1, respectively. Both intrinsic and extrinsic apoptotic pathway were identified in regulating the cell death caused by Ch-1 in Hela cells. p53, the upstream regulator of apoptotic pathway were extremely significantly up-regulated in Hela cells treated with 25 μM Ch-1. Moreover, the inhibiting effects and apoptotic related proteins responses to Ch-1 on Hela cells were abolished after pre-treated with Pifithrin-α (Pft-α), a p53 inhibitor. So, p53-depedent apoptosis is the crucial factor governing the inhibiting effects of Ch-1 in Hela cells. Amazingly, Ch-1 at non-toxic concentration (2.5–10 μM) enhanced significantly anti-cancer effect of 10-hydroxy camptothecin (HCPT) on Hela, BGC823, and MCF-7 cells.     

The defensin from avocado (Persea americana var. drymifolia) PaDef induces apoptosis in the human breast cancer cell line MCF-7

Antimicrobial peptides (AMPs) are cytotoxic to cancer cells; however, mainly the effects of AMPs from animals have been evaluated. In this work, we assessed the cytotoxicity of PaDef defensin from avocado (Persea americana var. drymifolia) on the MCF-7 cancer cell line (a breast cancer cell line) and evaluated its mechanism of action. PaDef inhibited the viability of MCF-7 cells in a concentration-dependent manner, with an IC₅₀ = 141.62 μg/ml. The viability of normal peripheral blood mononuclear cells was unaffected by this AMP. Additionally, PaDef induced apoptosis in MCF-7 cells in a time-dependent manner, but did not affect the membrane potential or calcium flow. In addition, PaDef IC₅₀ induced the expression of cytochrome c, Apaf-1, and the caspase 7 and 9 genes. Likewise, this defensin induced the loss of mitochondrial Δψm and increased the phosphorylation of MAPK p38, which may lead to MCF-7 apoptosis by the intrinsic pathway. This is the first report of an avocado defensin inducing intrinsic apoptosis in cancer cells, which suggests that it could be a potential therapeutic molecule in the treatment of cancer.     

The reduction of IL-6 gene expression,pAKT, pERK1/2, pSTAT3 signaling pathways and invasion activity by gallic acid in prostate cancer PC3 cells

Prostate cancer (PC) is one of the most common cancers among men. Progression of prostate cancer is associated with an increase in cellular level of interleukin-6 (IL-6). Gallic acid (GA) is a polyhydroxy phenolic compound which can inhibit the growth of cancer cells. The aim of this study was to evaluate the effects of GA treatment on cell viability, proliferation, invasion, IL-6 gene expression, IL-6 secretion, cellular levels of pSTAT3, pERK1/2, and pAKT signaling proteins in human prostate cancer PC3 cells. PC3 cells viability after treatment with GA (0–120 μM) was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The expression of IL-6 was investigated using real-time polymerase chain reaction. Cellular concentration of pSTAT3, pERK1/2, and pAKT signaling proteins were determined by Western blotting technic. PC3 cells invasion was assessed by invasion assay test. Treatment with GA caused a significant decrease in cell viability, proliferation, invasion, cellular levels of pSTAT3, pERK1/2, and pAKT signaling proteins after 48 h in a dose-dependent manner. The level of IL-6 and its gene expression decreased significantly in PC3 cells treated with GA. Our results show that IL-6 down-regulation and decreased IL-6 protein level in PC3 cells by GA resulted in diminishing of pSTAT3, pERK1/2, and pAKT signaling proteins which lead to the reduction of the cell survival, proliferation, and invasion in PC3 cells. Therefore, it seems that GA can be considered an anticancer agent in the treatment of prostate cancer.     

Interferences of homogentisic acid (HGA) on routine clinical chemistry assays in serum and urine and the implications for biochemical monitoring of patients with alkaptonuria

ObjectivesWe have assessed the effect of elevated concentrations of homogentisic acid (HGA) as in alkaptonuria (AKU), on a range of routine chemistry tests in serum and urine.Design and methodsHGA was added to pooled serum and a range of assays was analysed with Roche Modular chemistries. Effects on urine were assessed by diluting normal urine with urine from a patient with AKU, adding HGA to urine and after lowering output of urinary HGA with nitisinone treatment.ResultsSerum enzymatic creatinine showed 30% negative interference with 100 μmol/L HGA and > 50% at 400 μmol/L. Serum urate 100 to 480 μmol/L was reduced up to 20% at 100 and to 50% with 400 μmol/L HGA. Serum cholesterol between 3 and 11 mmol/L was reduced by 0.5 mmol/L with 400 μmol/L HGA. Urine enzymatic creatinine and urate with > 2 mmol/L HGA showed concentration dependent negative interference up to 80%. A positive interference in urine total protein by benzethonium turbidometric assay was observed, with 10 mmol/L HGA equivalent to 1 g/L protein. Jaffe creatinine, Na, K, Cl, Mg, Ca, phosphate, ALT, GGT, ALP activities and urea in serum and or urine were not affected by increases in HGA.ConclusionsTo avoid interferences by HGA in alkaptonuria concentration of HGA should be established before samples are assayed with peroxidase assays and benzethonium urine protein.     

The expression of TSSC3 and its prognostic value in patients with osteosarcoma

Osteosarcoma is one of the most common primary bone tumors in children and adolescents, typically presenting with poor prognosis. Recent studies have found that TSSC3 had a potential capability in suppressing the tumor development in osteosarcoma. Our purpose was to explore the role of TSSC3 in the clinical outcome of osteosarcoma patients. Firstly, we detected the expression of TSSC3 at mRNA level by quantitative real-time polymerase chain reaction (qRT-PCR). The result demonstrated that TSSC3 expression was lower in osteosarcoma patients than in healthy controls (P < 0.05). Then, the relationship between TSSC3 and clinicopathological characteristics was analyzed by chi-square test which manifested that WHO grade, metastasis, and recurrence were vital influential factors on the expression of TSSC3 (P < 0.05). We also estimated the association between TSSC3 and overall survival of osteosarcoma patients by Kaplan–Meier analysis as well as assessed the prognostic value of TSSC3 and clinicopathological characteristics through cox regression analysis. Patients with high TSSC3 expression were proved to live longer than those with low TSSC3 expression (log rank test, P < 0.05). TSSC3 expression (P = 0.032, HR = 0.405, 95%CI = 0.177–0.926) and metastasis (P = 0.010, HR = 2.849, 95%CI = 1.291–6.287) were considered to be independent prognostic factors in osteosarcoma. Taken together, our findings provided preliminary evidence that the TSSC3 was a prognostic marker in osteosarcoma and this might be useful for the therapy of osteosarcoma.     

Superiority of postmortem liver fentanyl concentrations over peripheral blood influenced by postmortem interval for determination of fentanyl toxicity

ObjectiveThe current study was undertaken to determine the relationship between postmortem (PM) peripheral blood (PB) and liver fentanyl concentrations and the role of measuring liver fentanyl concentrations in cause of death investigations in medical examiner cases in which fentanyl was identified.Design and methodsFB and liver tissue were routinely collected at autopsy from 4 Minnesota medical examiners' offices in 2010–2011. Samples were analyzed by gas chromatography–mass spectrometry (GC–MS).ResultsPB fentanyl ranged from < 2–15 μg/L in non-drug related deaths (n = 5), < 2–22 μg/L from mixed drug toxicity (n = 26) and 3.7–56 μg/L from fentanyl toxicity (n = 33). Liver fentanyl ranged from 11 to 104 μg/kg, 6 to 235 μg/kg, and 18 to 365 μg/kg, respectively. PB and liver fentanyl showed a modest correlation (r = 0.67). PM interval to the liver/blood ratio showed a decreasing ratio over increasing PM interval in cases from fentanyl and mixed drug toxicity. Liver fentanyl concentrations best define therapeutic use at < 23 μg/kg and fatal toxicity at > 56 μg/kg, without substantial overlap as found in blood fentanyl concentrations.ConclusionDiscriminatory liver fentanyl concentrations suggestive of therapeutic or toxic drug levels may better assist cause of death determination in cases of suspected fentanyl toxicity than postmortem PB concentrations. Peripheral blood fentanyl concentrations appear to undergo postmortem redistribution, associated with an increasing PM interval.     

Alterations of Na+/K+-ATPase,cholinergic and antioxidant enzymes activity by protocatechuic acid in cadmium-induced neurotoxicity and oxidative stress in Wistar rats

BackgroundThis study assessed the possible protective mechanisms of protocatechuic acid (PCA) against cadmium (Cd)-induced oxidative stress and neurotoxicity in rats.MethodsMale wistar strain rats weighing between 150–160 g were purchased and acclimatized for two weeks. The rats were divided into seven groups of seven each; NC group received normal saline, CAD group received 6 mg/kg of Cd-solution, CAD + PSG group received Cd-solution and prostigmine (5 mg/kg), CAD + PCA-10 and CAD + PCA-20 groups received Cd-solution and PCA (10 mg/kg and 20 mg/kg) respectively, PCA-10 and PCA-20 groups received 10 mg/kg and 20 mg/kg PCA each. Animals were administered normal saline, Cd and PCA daily by oral gavage for 21 days. After which the animals were sacrificed, the brain excised, homogenized and centrifuged. The activities of enzymes (Na⁺/K⁺-ATPase, cholinesterases, catalase, glutathione peroxidase, superoxide dismutase) and levels of oxidative stress markers (lipid peroxidation and reduced glutathione) linked to neurodegeneration were subsequently assessed.ResultsSignificant (p < 0.05) alterations in the enzyme activities and levels of oxidative stress markers were observed in CAD group when compared to the NC group. However, the activities of the enzymes were reversed in CAD + PSG and CAD + PCA groups.ConclusionsPCA may protect against cadmium-induced neurotoxicity by altering the activities of Na⁺/K⁺-ATPase, acetylcholinesterase, butyrylcholinesterase and endogenous antioxidant enzymes.     

Exenatide suppresses 1,2-dimethylhydrazine-induced colon cancer in diabetic mice: Effect on tumor angiogenesis and cell proliferation

Colon cancer is the third leading cause of cancer mortality worldwide, which results from interactions of different factors. It is frequently a pathological consequence of persistent inflammation. Diabetes affects several cancers and is positively correlated with the incidence of colon cancer. This study aimed to study the effect of exenatide in ameliorating inflammation, angiogenesis and cell proliferation in 1,2-dimethyl hydrazine (DMH) induced colorectal carcinoma in diabetic mice. Mice were randomly allocated into six groups, 8 mice each. Group 1: vehicle control group. Group 2: diabetic control group. Group 3: DMH control group: diabetic mice treated with DMH (20 mg/kg/week, s.c.) for 15 week. Group 4: DMH-cisplatin group: mice received cisplatin (4 mg/kg/week, i.p.). Groups 5 & 6: DMH-exenatide (10 and 20 μg/kg) group: mice received exenatide (10 or 20 μg/kg/day, s.c.), respectively. The present results highlighted an increase in angiogenic markers and cell proliferation in the DMH-diabetic group in comparison with the control group with greater expression of endothelial marker (CD₃₄) and Ki-67 in colon tissue. Monotherapy with cisplatin or exenatide (10 and 20 μg/kg) downregulated these markers to different extents. The current results provided evidence that exenatide represents a promising chemopreventive effect against DMH-induced colon carcinogenesis in diabetic mice, at least in part, attributed to its anti-angiogenic and anti-proliferative mechanisms.     

Effects of chlorogenic acid on adenine nucleotides hydrolyzing enzyme activities and expression in platelets of rats experimentally demyelinated with ethidium bromide

BackgroundThe effects of chlorogenic acid (one of the major phenolic acid found in human diets) were investigated on the adenine nucleotides hydrolyzing enzymes; ecto-nucleotide pyrophosphatase/phophodiesterase (E-NPP), ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase), E-5′- nucleotidase and ecto-adenosine deaminase (E-ADA) activities and expression in platelets of rats experimentally demyelinated with ethidium bromide.MethodsRats were divided into four groups of eight animals each. Group I rats were control rats; injected with saline (CT), group II rats were injected with saline and treated with chlorogenic acid (AC), group III rats were injected with 0.1% ethidium bromide (EB) and group IV rats were injected with 0.1% EB and treated with chlorogenic acid (EB + AC). The activities of the enzymes were analyzed using colorimetric methods, and the gene expression of NTPDase 1, 2 and 3 were analyzed using the polymerase chain reaction (PCR).ResultsThe results revealed that there was a significant (P < 0.01) reduction in E-NPP activity in EB group (1.63 ± 0.10 nmol p-nitrophenol released/min/mg protein) when compared to CT group (2.33 ± 0.14 nmol p-nitrophenol released/min/mg protein). However, treatment with chlorogenic acid significantly (P < 0.05) increased E-NPP activity in EB group. Furthermore, no significant (P > 0.05) change was observed in the E-NPP activity of EB + AC group (2.19 ± 0.08 nmol p-nitrophenol released/min/mg protein) when compared to CT group (2.33 ± 0.14 nmol p-nitrophenol released/min/mg protein). In addition, there was a significant (P < 0.05) increase in AMP hydrolysis in EB rat group when compared to CT group. No significant (P > 0.05) difference was observed in AMP hydrolysis between AC, AC + EB and CT groups. Conversely, there were no significant (P > 0.05) differences in ATP and ADP hydrolyses between all the groups (AC, EB, AC + EB and CT groups). Likewise, there were no significant (P > 0.05) changes in E-ADA activity and percentage platelet aggregation among all groups studied. Similarly, no significant (P > 0.05) change was observed in the expression of E-NTPDase 1, 2 and 3 in all the groups tested.ConclusionsOur study revealed that chlorogenic acid may modulate the hydrolysis of adenine nucleotides in platelets of rats demyelinated and treated with chlorogenic acid via alteration of E-NPP and ecto-5′-nucleotidase activities.