Functional assays to determine the significance of two common XPC 3'UTR variants found in bladder cancer patients

<p>Abstract</p> <p>Background</p> <p><it>XPC </it>is involved in the nucleotide excision repair of DNA damaged by carcinogens known to cause bladder cancer. Individuals homozygous for the variant allele of <it>XPC </it>c.1496C > T (p.Ala499Val) were shown in a large pooled analysis to have an increased bladder cancer risk, and we found two 3'UTR variants, *611T > A and c.*618A > G, to be in strong linkage disequilibrium with c.1496T. Here we determined if these two 3'UTR variants can affect mRNA stability and assessed the impact of all three variants on mRNA and protein expression.</p> <p>Methods</p> <p><it>In vitro </it>mRNA stability assays were performed and mRNA and protein expression measured both in plasmid-based assays and in lymphocytes and lymphoblastoid cell lines from bladder and breast cancer patients.</p> <p>Results</p> <p>The two 3'UTR variants were associated with reduced protein and mRNA expression in plasmid-based assays, suggesting an effect on mRNA stability and/or transcription/translation. A near-significant reduction in XPC protein expression (p = 0.058) was detected in lymphoblastoid cell lines homozygous for these alleles but no differences in mRNA stability in these lines was found or in mRNA or protein levels in lymphocytes heterozygous for these alleles.</p> <p>Conclusion</p> <p>The two 3'UTR variants may be the variants underlying the association of c.1496C > T and bladder cancer risk acting via a mechanism modulating protein expression.</p

In vitro functional effects of XPC gene rare variants from bladder cancer patients

The XPC gene is involved in repair of bulky DNA adducts formed by carcinogenic metabolites and oxidative DNA damage, both known bladder cancer risk factors. Single nucleotide polymorphisms (SNPs) in XPC have been associated with increased bladder cancer risk. Recently, rarer genetic variants have been identified but it is difficult to ascertain which are of functional importance. During a mutation screen of XPC in DNA from 33 bladder tumour samples and matched blood samples, we identified five novel variants in the patients’ germ line DNA. In a case–control study of 771 bladder cancer cases and 800 controls, c.905T>C (Phe302Ser), c.1177C>T (Arg393Trp), c.*156G>A [3′ untranslated region (UTR)] and c.2251-37C>A (in an intronic C>G SNP site) were found to be rare variants, with a combined odds ratio of 3.1 (95% confidence interval 1.0–9.8, P = 0.048) for carriage of one variant. The fifth variant was a 2% minor allele frequency SNP not associated with bladder cancer. The two non-synonymous coding variants were predicted to have functional effects using analytical algorithms; a reduced recruitment of GFP-tagged XPC plasmids containing either c.905T>C or c.1177C>T to sites of 408 nm wavelength laser-induced oxidative DNA damage was found in vitro. c.*156G>A appeared to be associated with reduced messenger RNA stability in an in vitro plasmid-based assay. Although the laser microbeam assay is relevant to a range of DNA repair genes, our 3′ UTR assay based on Green fluorescent protein(GFP) has widespread applicability and could be used to assess any gene. These assays may be useful in determining which rare variants are functional, prior to large genotyping efforts

Lead compounds for prion therapeutics from Chinese herbs

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A mass spectrometry guided elution-extrusion counter-current chromatography protocol for isolation of eighteen terpenoids from Fructus Corni and assessment of their anti-glioma activities

The utilization of counter-current chromatography (CCC) requires the assistance of the analytical instruments to measure the distribution constants (KC) values and to monitor the CCC running process. Due to the limitation of the conventional detection means, CCC is not ideal for isolating low-concentration components which are most likely to be new compounds. We introduced ion current extraction (ICE), a MS-based data post-acquiring method, to be an auxiliary tool to prominently boost CCC separation power and scope in the current work. An elution-extrusion CCC protocol was set up. For the first time, ICE was applied to process LC-MS and CCC-MS data so that extracted ion currents (EICs) of target c"onstituents were generated. In the obtained LC-EIC and CCC-EIC chromatograms obtained, the target peaks were reinforced and the chemical noise was diminished, which facilitated the KC values determination and the target compounds fractionation. Eighteen terpenoids including a new compound were isolated from Fructus Corni. Their bioactivities were tested in glioma cell U87 and LN229, which revealed remarkable inhibiting effects from triterpenoids in this plant. The proposed CCC strategy showed potential to discover, analyze, trace and fractionate chemicals, especially minor ones that are possibly novel or bioactive compounds, from a complex sample

Aquaporins Alteration Profiles Revealed Different Actions of Senna, Sennosides, and Sennoside A in Diarrhea-Rats

Senna and its main components sennosides are well-known effective laxative drugs and are used in the treatment of intestinal constipation in the world. Their potential side effects have attracted more attention in clinics but have little scientific justification. In this study, senna extract (SE), sennosides (SS), and sennoside A (SA) were prepared and used to generate diarrhea rats. The diarrhea rats were investigated with behaviors, clinical signs, organ index, pathological examination, and gene expression on multiple aquaporins (Aqps) including Aqp1, Aqp2, Aqp3, Aqp4, Aqp5, Aqp6, Aqp7, Aqp8, Aqp9, and Aqp11. Using qRT-PCR, the Aqp expression profiles were constructed for six organs including colon, kidney, liver, spleen, lung, and stomach. The Aqp alteration profiles were characterized and was performed with Principle Component Analysis (PCA). The SE treatments on the rats resulted in a significant body weight loss (p &lt; 0.001), significant increases (p &lt; 0.001) on the kidney index (27.72%) and liver index (42.55%), and distinguished changes with up-regulation on Aqps expressions in the kidneys and livers. The SS treatments showed prominent laxative actions and down regulation on Aqps expression in the colons. The study results indicated that the SE had more influence/toxicity on the kidneys and livers. The SS showed more powerful actions on the colons. We suggest that the caution should be particularly exercised in the patients with kidney and liver diseases when chronic using senna-based products

Imatinib Radiosensitizes Bladder Cancer by Targeting Homologous Recombination

Radiotherapy is a major treatment modality used to treat muscle-invasive bladder cancer, with patient outcomes similar to surgery. However, radioresistance is a significant factor in treatment failure. Cell-free extracts of muscle-invasive bladder tumors are defective in nonhomologous end-joining (NHEJ), and this phenotype may be used clinically by combining radiotherapy with a radiosensitizing drug that targets homologous recombination, thereby sparing normal tissues with intact NHEJ. The response of the homol- ogous recombination protein RAD51 to radiation is inhibited by the small-molecule tyrosine kinase inhibitor imatinib. Stable RT112 bladder cancer Ku knockdown (Ku80KD) cells were generated using short hairpin RNA technology to mimic the invasive tumor phenotype and also RAD51 knockdown (RAD51KD) cells to show imatinib's pathway selectivity. Ku80KD, RAD51KD, nonsilencing vector control, and parental RT112 cells were treated with radiation in combination with either imatinib or lapatinib, which inhibits NHEJ and cell survival assessed by clonogenic assay. Drug doses were chosen at approximately IC40 and IC10 (nontoxic) levels. Imatinib radiosensitized Ku80KD cells to a greater extent than RAD51KD or RT112 cells. In contrast, lapatinib radiosensitized RAD51KD and RT112 cells but not Ku80KD cells. Taken together, our findings suggest a new application for imatinib in concurrent use with radiotherapy to treat muscle-invasive bladder cance

Neuroprotective effect of Apelin 13 on ischemic stroke by activating AMPK/GSK-3β/Nrf2 signaling

Abstract Background Previous studies had showed that Apelin 13 could protect against apoptosis induced by ischemic/reperfusion (I/R). However, the mechanisms whereby Apelin 13 protected brain I/R remained to be elucidated. The present study was designed to determine whether Apelin 13 provided protection through AMPK/GSK-3β/Nrf2 pathway. Methods In vivo, the I/R model was induced and Apelin 13 was given intracerebroventricularly 15 min before reperfusion. The neurobehavioral scores, infarction volumes, and some cytokines in the brain were measured. For in vitro study, PC12 cells were used. To clarify the mechanisms, proteases inhibitors or siRNA were used. Protein levels were investigated by western blotting. Results The results showed that Apelin 13 treatment significantly reduced infarct size, improved neurological outcomes, decreased brain edema, and inhibited cell apoptosis, oxidative stress, and neuroinflammation after I/R. Apelin 13 significantly increased the expression of Nrf2 and the phosphorylation levels of AMPK and GSK-3β. Furthermore, in cultured PC12 cells, the same protective effects were also observed. Silencing Nrf2 gene with its siRNA abolished the Apelin 13’s prevention of I/R-induced PC12 cell injury, oxidative stress, and inflammation. Inhibition of AMPK by its siRNA decreased the level of Apelin 13-induced Nrf2 expression and diminished the protective effects of Apelin 13. The interplay relationship between GSK-3β and Nrf2 was also verified with relative overexpression. Using selective inhibitors, we further identified the upstream of AMPK/GSK-3β/Nrf2 is AR/Gα/PLC/IP3/CaMKK. Conclusions In conclusion, the previous results showed that Apelin 13 protected against I/R-induced ROS-mediated inflammation and oxidative stress through activating the AMPK/GSK-3β pathway by AR/Gα/PLC/IP3/CaMKK signaling, and further upregulated the expression of Nrf2-regulated antioxidant enzymes

A mass spectrometry guided elution-extrusion counter-current chromatography protocol for isolation of eighteen terpenoids from Fructus Corni and assessment of their anti-glioma activities

The utilization of counter-current chromatography (CCC) requires the assistance of the analytical instruments to measure the distribution constants (KC) values and to monitor the CCC running process. Due to the limitation of the conventional detection means, CCC is not ideal for isolating low-concentration components which are most likely to be new compounds. We introduced ion current extraction (ICE), a MS-based data post-acquiring method, to be an auxiliary tool to prominently boost CCC separation power and scope in the current work. An elution-extrusion CCC protocol was set up. For the first time, ICE was applied to process LC-MS and CCC-MS data so that extracted ion currents (EICs) of target c"onstituents were generated. In the obtained LC-EIC and CCC-EIC chromatograms obtained, the target peaks were reinforced and the chemical noise was diminished, which facilitated the KC values determination and the target compounds fractionation. Eighteen terpenoids including a new compound were isolated from Fructus Corni. Their bioactivities were tested in glioma cell U87 and LN229, which revealed remarkable inhibiting effects from triterpenoids in this plant. The proposed CCC strategy showed potential to discover, analyze, trace and fractionate chemicals, especially minor ones that are possibly novel or bioactive compounds, from a complex sample