Targeting the anaphase-promoting complex/cyclosome (APC/C) enhanced antiproliferative and apoptotic response in bladder cancer

Improving the chemotherapy sensitivity of bladder cancer is a current clinical challenge. It is critical to seek out effective combination therapies that include low doses of cisplatin due to its dose-limiting toxicity. This study aims to investigate the cytotoxic effects of the combination therapy including proTAME, a small molecule inhibitor, targeting Cdc-20 and to determine the expression levels of several APC/C pathway-related genes that may play a role in the chemotherapy response of RT-4 (bladder cancer) and ARPE-19 (normal epithelial) cells. The IC20 and IC50 values were determined by MTS assay. The expression levels of apoptosis-associated (Bax and Bcl-2) and APC/C-associated (Cdc-20, Cyclin-B1, Securin, and Cdh-1) genes were assessed by qRT-PCR. Cell colonization ability and apoptosis were examined by clonogenic survival experiment and Annexin V/PI staining, respectively. Low-dose combination therapy showed a superior inhibition effect on RT-4 cells by increasing cell death and inhibiting colony formation. Triple-agent combination therapy further increased the percentage of late apoptotic and necrotic cells compared to the doublet-therapy with gemcitabine and cisplatin. ProTAME-containing combination therapies resulted in an elevation in Bax/Bcl-2 ratio in RT-4 cells, while a significant decrease was observed in proTAME-treated ARPE-19 cells. Cdc-20 expression in proTAME combined treatment groups were found to be decreased compared to their control groups. Low-dose triple-agent combination induced cytotoxicity and apoptosis in RT-4 cells effectively. It is essential to evaluate the role of APC/C pathway-associated potential biomarkers as therapeutic targets and define new combination therapy regimens to achieve improved tolerability in bladder cancer patients in the future

Comparison of DNA isolation methods from mammalian sperm cells and development of a new protocol

Sperm DNA tightly packed with protamines makes the DNA isolation procedure from sperm cells long and laborious. Cell lysis is also a challenging step because of the disulfide bonds-rich membranes of the sperm cells. In this study we aimed to evaluate potential rapid DNA isolation protocols to isolate DNA from mammalian sperm cells, and develop an easy, rapid, and cost-effective protocol for sperm DNA isolation which can be used in molecular biology and diagnostics. Sperm samples were collected from seven adult rats. Our developed protocol included Proteinase K and small amount of β-mercaptoethanol (βME) for cell lysis. A modified salting-out technique was then employed to collect DNA. Alternative protocols involving InstaGene matrix and cell sonication techniques were also applied to achieve DNA isolation. Concentration of the DNA yield was measured, and the degradation of DNA was checked using agarose gel electrophoresis. The intactness of the DNA yield was assessed and validated using polymerase chain reaction (PCR) and capillary gel electrophoresis techniques. The lysis of the cells and high-quality DNA yield have only been achieved using our developed optimized protocol. To confirm the quality of DNA for assays, PCR product was synthesized for rat actin β (RActβ) gene and then analyzed using capillary gel electrophoresis. A strong peak at right m/z value for the amplicon was obtained. We described an improved protocol over the previous methods suggesting the use of combined commercial kits and long incubation times

Investigation of a glioblastoma risk-associated SNP of the PTPRB Gene in familial glioblastoma

Objective: To investigate the association of the single nucleotide polymorphism (SNP) rs2252784 in the protein tyrosine phos-phatase receptor type B (PTPRB) gene with familial glioblastoma multiforme (GBM). Material and Methods: Genomic DNA was extracted from the peripheral blood samples of 2 sibling GBM patients, their 6 family members and 2 formalin-fixed paraffin-embedded (FFPE) tumor tissues. A 400 bp region was amplified and the restriction fragment length polymorphism (RFLP) technique was used to identify the rs2252784 SNP in exon 2 of the PTPRB gene. The GBM cell line T98G was used to validate the findings obtained from the tumor samples. Results: The analysis of DNA obtained from the blood samples of both GBM patients showed a wild-type (WT) genotype. However, the results of the PCR-RFLP analysis from FFPE tumor tissues showed that the first patient (proband) was heterozygous, and his sibling was homozygous for the rs2252784 variant. Discordant results between SNP analyses of the DNA samples isolated from the blood and FFPE tumor tissue were ob-served. The family members of the patients had either homozygous WT or heterozygous variants. Conclusion: The rs2252784 SNP was present in the tumor DNA of the patients but not in the DNA samples obtained from blood. This discrepancy might be the result of oncogenic DNA alterations associated with tumor formation. Paired analysis of tumors and blood samples from patients and patient-matched normal blood samples from GBM-affected families might provide additional insights into the underlying genetic alterations that occur during the development of a tumor in familial GBM

Anticancer effects of a novel herbal combination as a potential therapeutic candidate against lung cancer

Introduction: Cancer continues to be a prevalent health problem with increasing mortality rates. The interest in complementary medicine for cancer treatment has increased in recent years. In this study, we aimed to investigate whether there are in vitro anticancer effects of a commercially available polyherbal formulation (PHF) against lung cancer. Methods: MTS assay, colony formation, wound healing, Annexin-V/PI assays and qRT-PCR were performed to determine the effects of PHF on viability, colony forming ability, migration and apoptosis of non-small cell lung cancer (NSCLC) cells, A549 and human retinal epithelial cells, ARPE-19 as a non-cancerous control. Results: We examined the cytotoxic effect of PHF on cell proliferation in comparison with cisplatin and both showed significant cytotoxic effects on A549 cells. PHF treatment resulted in a relatively mild cytotoxic effect against ARPE-19 cells compared to cisplatin treatment. The inhibition of growth in more than 50% of A549 cells was noted with either 1:25 dilution of PHF or 24 mu M of cisplatin treatments. PHF caused a more effective suppression of cell migration compared to cisplatin (P<0.001). Both PHF and cisplatin at IC50 doses effectively inhibited colony formation and induced apoptosis. Bax/Bcl-2 ratio was elevated in A549 cells after both PHF and cisplatin treatments. Conclusion: Our data indicate that PHF might have potent migration inhibitory and apoptosis-inducing activities. More importantly, PHF showed a less cytotoxic effect on normal cells than cisplatin. The mechanisms underlying the anticancer activity of PHF need to be elucidated more comprehensively to confirm its potential as a promising therapeutic candidate

Repurposing ibudilast in combination with temozolomide for glıoblastoma

21st Annual Scientific Meeting and Education Day of the Society-for-Neuro-Oncology -- NOV 17-20, 2016 -- Scottsdale, AZWOS: 000398604101198Aims: Recurrence in patients with Glioblastoma (GBM) is inevitable, even in patients with O-6-Methylguanine-DNA Methyl Transferase (MGMT) methylation.Soc Neuro Onco

To determine LDL phenotypes using lipids, lipoproteins, apoproteins, and sdLDL through association rule mining

Objective: The atherogenic lipoprotein phenotype is closely associated with the risk assessment of Coronary Artery Disease (CAD) and the monitoring of treatment processes. Particularly, high levels of small dense low-density lipoprotein (sdLDL) and low levels of large buoy-ant low-density lipoprotein (lbLDL) are critical in determining Pattern B. This study aims to determine the lipid phenotype using the Association Rule Mining (ARM) method, based on concentrations of lipids, lipoproteins, apoproteins, and sdLDL.Materials and Methods: This retrospective case-control study utilized analytical research methods. Numerical variables were expressed as mean, standard deviation, median, and min-max values. Statistically significant differences were observed between the low-density lipoprotein (LDL) size categories in terms of triglycerides (TG), LDL, high-density lipoprotein (HDL), apolipoprotein B (ApoB), apolipoprotein E (ApoE), sdLDL, and lbLDL distributions. ARM was employed to detect the lipoprotein phenotype.Results: Statistically significant differences were found between the LDL size categories in distributions of TG, LDL, HDL, ApoB, ApoE, sdLDL, and lbLDL (p(TG)<0.001, p(LDL)=0.03, p(HDL)<0.001, p(ApoB)=0.016, p(ApoE)=0.004, p(sdLDL)<0.001, and p(lbLDL)<0.001). The ARM method revealed that the probability of phenotype B is 100% for sdLDL values in the range of 15.5-109 and lbLDL values in the range of 0-31.5.Conclusion: This study introduces a contemporary approach for detecting lipoprotein phenotypes using ARM, further substantiating the strong correlation between atherogenic phenotypes and sdLDL

The association of Sort1 expression with LDL subfraction and inflammation in patients with coronary artery disease

Background: Controversial effect of sortilin on lipoprotein metabolism in the development of atherosclerosis reveals the need for more extensive research Objectives: The aim of this study was to investigate the association between Sort1 gene expression and lipids, lipoprotein subfractions, and inflammation in CAD. Methods: The study population included 162 subjects with CAD and 49 healthy individuals. The Sort1 gene expression level was determined by qRT-PCR using Human Sortilin TaqMan Gene Expression Assays. Lipoprotein subclasses were analysed by the Lipoprint system. Serum levels of apolipoprotein and CRP were measured by autoanalyzer. Results: Sort1 gene expression and atherogenic subfraction (SdLDL) levels were significantly higher (p < 0.001) while atheroprotective subfraction (LbLDL) was lower in the subjects with CAD (p < 0.050). Also, increased Sort1 gene expression levels were observed in those with higher CRP values. Conclusions: Our findings reveal that the high Sort1 gene expression has a prominent linear relationship with both the atherogenic LDL phenotype and proinflammation, thereby might contribute to the occurrence of CA

Development of MoS2 and Au nanoparticle including disposable CEA-based immuno-cytosensor platforms

In this study, an electrochemical biosensor for determination of Carcinoembryonic antigen (CEA) biomarker using human breast ductal adenocarcinoma (MCF-7) and human ovarian adenocarcinoma (SK-OV-3) cancer cells was presented. Disposable pencil graphite electrodes (PGE) has been modified with Au nanoparticle (Au NP) and MoS2 nanostructure dispersed chitosan (Cs) matrix. Therefore, the electrochemical interaction between the antibody-electrode surface was facilitated. Under optimal conditions, the immunosensor exhibited high sensitivity toward CEA biomarker in the low concentration range 0.01-10 ng mL(-1), with the detection limit of 1.93 ng mL(-1) and relative standard deviation of 4.65 (n = 5). The results indicated that even very small changes in CEA concentration can be sensed with the presented system. Also, recovery of the immunosensor found as 98 +/- 3% in the real sera samples containing dopamine and ascorbic acid. It has a great potential in the clinical screening of divergent cancer biomarkers. Experiments were conducted to determine the number of normal cell hGF and cancer cells adhered and attached on immuno-cytosensor surfaces. CEA-positive MCF-7 cells have shown great potential for adhesion and attachment to MoS2/Cs/Au/Anti-CEA/CEA surface better than CEA-negative cells. The developed immuno-cytosensor exhibited very promising results for the future biosensor studies

Prevention of carbon tetrachloride- induced Hepatotoxicity by Urtica urens in rats

In this study, the effects of Urtica urens L. (dwarf nettle, UU) seed extract on lipid peroxidation, antioxidant and xenobiotic metabolizing enzymes in both control and carbon tetrachloride (CCl4)-treated rats were investigated. Male Wistar rats were randomly allotted into one of the four experimental groups. A (Control), B (UU-treated), C (CCl4-only treated) and D (UU+CCl4-treated), each having 5-24 animals. Some of rats in group A were treated with physiological saline, i.p. daily for 4 days; Group B were treated with UU 200 mg/kg, i.p. daily for 4 consecutive days; Group C were administered with CCl4 at dose of 10 ml/kg, i.p. for 2 consecutive days; and group D rats were pretreated with UU 200 mg/kg, i.p. daily for 4 consecutive days prior to administration of CCl4 10 ml/kg, i.p. daily for 2 consecutive days. At the end of the experimental period, rats were sacrificed, and tissues were taken. Results have indicated that treatment of rats with U. urens increased hepatic antioxidant enzymes without changing the levels of serum Lactate DeHydrogenase (LDH), ALanine aminoTransferase (ALT) and ASpartate aminoTransferase (AST). Moreover, U. urens treatment decreased the CCl4 dependent elevated lipid peroxidation and serum LDH, ALT and AST activities. Furthermore, U. urens protected the inhibitory effect of CCl4 on CYP2E1 catalyzed aniline 4-hydroxylase activities. As a result, as indicated by these in vivo data, U. urens seed extract contains constituents protecting liver against hepatotoxic effects of CCl4

Effects of nimesulide on the small intestine mucositis induced by methotrexate in rats

Arslan, Aynur/0000-0001-5968-5823; Cetin, Nihal/0000-0003-3233-8009;WOS: 000387526400001PubMed: 27333839Intestinal mucositis is one of the major problems in the patients receiving cancer treatment. Nimesulide is a drug with antioxidant, antiinflammatory and antiulcer features. We aimed to investigate the effect of nimesulide on the small intestine mucositis induced by methotrexate (MTX) in rats. Experimental animals were divided into the control group, MTX group (MTXG) and nimesulide+MTX administered group (NMTXG) with eight rats per group. the control and MTXG groups were given distilled water by gavage and the NMTXG was given nimesulide 100 mg/kg orally. After one hour, the NMTXG and MTXG rat groups were administered oral MTX 5 mg/kg. This procedure was repeated once a day for 15 days and the rats were sacrificed. the duodenum and jejunum of each rat was removed for the assessment of biochemical markers and histopathological evaluation. Malondialdehyde (MDA) and myeloperoxidase (MPO) levels were significantly higher in the duodenal and jejunal tissues of the animals which received MTX, compared to the control and NMTXG (P<0.001). Also, the levels of total glutathione (tGSH), glutathione reductase (GSHRd), glutathione peroxidase (GSHPx), catalase (CAT) and superoxide dismutase (SOD) were significantly lower in the MTXG (P<0.001) compared to other groups. MTX led to villus and crypt epithelial damage and inflammation containing marked PMNL and eosinophils in the intestinal tissues histopathologically. Whereas, there was only mild irregularities in the villus structures of the NMTXG. Nimesulide protected the small intestines against damage by MTX. Intestinal mucositis caused by MTX may be preventable by co-administered nimesulide