Elucidation of the WNT & AKT/Phosphoinositide-3-Kinase Pathways in Colorectal Carcinoma

Colorectal cancer (CRC) is the third most common cancer III Malaysia and is currently the commonest cancer in males. Genetics, experimental and epidemiological data suggest that CRC develops from complex interaction between inherited susceptibility and environmental factors. Accumulating evidence suggests that the Wnt and PI3K (phosphoinositide-3-kinase)/Akt signalling pathways playa causative role in tumorigenesis of colorectal cancer. By employing immunohistochemical method, the expressIOn and correlation of several key regulators or related biomolecules of the Wnt and PI3K1Akt signalling pathways in 47 archival formalin fixed, paraffin embedded tissues of surgically resected colorectal cancer (CRC) specimens performed at Kuala Lumpur Hospital (KLH) between 1999 and 2000, were studied. Laser captured microdissection technique, polymerase chain reaction and direct sequencing were used to investigate mutations in exon 3 of the p-catenin gene. Mutations in the mutation cluster region (MCR) of adenomatous polyposis coli (APC) gene were also investigated. The expressions of Wnt-l, WISP-l and FRAT-l mRNA were determined by reverse-transcription and real-time polymerase chain reaction method. The results showed that: The expressions ofWnt-l, FRAT-I, APC, nuclear p-catenin, cytoplasmic p-catenin, membrane p-catenin, membrane E-cadherin, cytoplasmic E-cadherin, WISP-I, cyclin-Dl, p-Aktl (Ser473), p-Akt1l2/3 (Thr308), p-BAD (Ser136), p-GSK 3p(Ser9) and survivin were found in 55.3%, 36.2%, 51.1% 44.6%, 95.7%,30.6%,46.8%,95.7%,31.9%, 10.6%,34%,44.7%,57.4% 44.7% and 59.6% of CRC tissues, respectively and 17.5%, 5% 100%, 0%, 75%, 100%, 100%, 50%, 12.5%, 0%, 5%, 12.5%, 22.5%, 22.5% and 32.5% of apparently normal adjacent tissues, respectively. The sum of scores for all biomolecules except APC, membrane p-catenin and membrane E-cadherin staining was significantly higher in CRC tissues in comparison to apparently normal adjacent tissues (p < 0.05). The sum of score for APC, membrane p-catenin and membrane E-cadherin staining was significantly lower in CRC tissues in comparison to apparently normal adjacent tissues (p < 0.05). The expression of Wnt and PI3K1Akt signalling pathway-related biomolecules was interrelated. The results ofnucleotide sequencing showed that no mutations at exon-3 of p-catenin were found. However, point mutations in the mutation cluster region of the APC gene leading to the formation of truncated APC protein, were found in four out eleven CRC tissues examined. A 1.43 to 21.26-foid and 1.11 to 109.14-fold increase in the level of expression of Wnt-l and FRAT-1 mRNA was found in eight out of eleven CRC tissues relative to apparently normal adjacent tissues. On the other hand, a 1.94 to 46.69-fold increase in the level of WISP-I mRNA was found in all the CRC tissues. This study has provided important information for researchers and clinicians in terms of clinical evidence of the involvement of the Wnt signalling pathway and PI3K1Akt signalling pathway in colorectal tumorigenesis. In addition, the present study also provided crucial information on the elucidation of the relationship between the biomolecules of these signalling pathways towards understanding their roles in colorectal tumourigenesis and the identification of potential targets for advance therapeutic intervention ofCRe. Based on our current results, we propose that Wnt-I, FRAT-1 and WISP-I could be served as potent therapeutic target for the treatment of CRe. On the basis of our present study, we conclude that the Wnt and PI3K1Akt signalling pathways are involved in tumourigenesis of CRC in Malaysia. These pathways are interrelated although they might also act independently in promoting tumour growth and inhibition of apoptosis. This study has also provided useful information for the search or design of better antitumour interventions

Characterisation of Plant Derived Damnacanthal and Nordamnacantbal Induced Cytotoxicity on Human HT29 Colon Adenocarcinoma Cell Line

Nordamnacanthal and damnacanthal are two anthraquinones isolated from the roots of Morinda elliptica. They were found to exhibit cytotoxic activity against HT29 human colon adenocarcinoma cells. The cytotoxic concentrations of damnacanthal and nordamnacanthal that inhibited 50% growth (IC₅₀) of HT29 were 17 µg/ml and 7 µg/ml respectively. For the comparative purposes, the ICsos of several cytotoxic drugs against HT29 were also determined. The inhibition effect of nordamnacanthal was found to be comparable to etoposide (IC₅₀ = 7 µg/ml). cisplatin (IC₅₀ = 5 µg/ml) and doxorubicin (IC₅₀ = 6 µg/ml). The compound was found to be less active than methotrexate (MTX) (IC₅₀ < 0.05 µg/ml) and leunase (IC₅₀ = 2 µg/ml). On the other hand, the cytotoxic effect of damnacanthal was less active as compared to all cytotoxic drugs. However both compounds were found to be less toxic against non-cancerous fibroblast 3T3 ceJJs with the ICsos of 30 /lglml (damnacanthal) and 21 /lglm] (nordamnacanthal) respectively. Furthermore, damnacanthal and nordamnacanthal were found to induce apoptosis on HT29 cells at their ICso concentration as demonstrated by conventional agarose gel electrophoresis and also morphological alterations. DNA laddering was obtained after 12 hours of treatment by both compounds in a dose-independent but time-dependent fashion. Both compounds also caused cell death with apoptotic features such as cell shrinkage, membrane blebbing, nuclear fragmentation, and the presence of apoptotic bodies. In addition, caspase-3 was found to be activated during the execution of apoptosis induced by these compounds. This caspase activation was inhibited by a peptide based general caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (Z-V AD-FMK). In conclusion, this study demonstrates the potential antitumor activites of damnacanthal and nordamnacanthal

Lens status influences the association between CFH polymorphisms and age-related macular degeneration: Findings from two population-based studies in Singapore

10.1371/journal.pone.0119570PLoS ONE103e011957

Association of Common SIX6 Polymorphisms With Peripapillary Retinal Nerve Fiber Layer Thickness: The Singapore Chinese Eye Study

PURPOSE. Recently the common SIX6 missense variant rs33912345 was found to be highly associated with glaucoma. The aim of this study was to investigate the association between this SIX6 variant and peripapillary retinal nerve fiber layer (RNFL) thickness measured by spectral-domain optical coherence tomography (SD-OCT) in a population setting. METHODS. Study subjects were enrolled from the Singapore Chinese Eye Study (SCES), a population-based survey of Singaporean Chinese aged 40 years or older. Subjects underwent a comprehensive ocular examination. Spectral-domain OCT was used to measure RNFL thicknesses. Genotyping of SIX6 rs33912345 (Asn141His) was performed using HumanExome BeadChip. RESULTS. A total of 2129 eyes from 1243 SCES subjects (mean age: 55.0 6 7.4 years) with rs33912345 genotype data and SD-OCT images were included for the analysis. Of these, 26 eyes of 21 subjects had glaucoma. The frequency of rs33912345 risk variant C (His141) was 80% in the study subjects. Each rs33912345 C allele was associated with a decrease of 1.44 lm in RNFL thickness after adjusting for age, sex, genetic principal components, and axial length (P ¼ 0.001). These associations remained similar in 2096 nonglaucoma eyes in which each C allele was associated with a decrease of 1.39 lm in RNFL thickness (P ¼ 0.001). The strongest association was observed in the superior RNFL sector (a decrease of 2.83 lm per risk allele, P &lt; 0.001) followed by the inferior RNFL sector (a decrease of 2.24 lm per risk allele, P ¼ 0.003), while the association did not reach significance in the nasal and temporal sectors. CONCLUSIONS. Nonglaucomatous individuals with the SIX6 missense variant have reduced RNFL thickness in regions known to be particularly affected in those with glaucoma. This may be the primary mechanism for increased risk of POAG in individuals who carry the SIX6 His141 risk variant

Nrf2 Expression Is Regulated by Epigenetic Mechanisms in Prostate Cancer of TRAMP Mice

Nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) is a transcription factor which regulates the expression of many cytoprotective genes. In the present study, we found that the expression of Nrf2 was suppressed in prostate tumor of the Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) mice. Similarly, the expression of Nrf2 and the induction of NQO1 were also substantially suppressed in tumorigenic TRAMP C1 cells but not in non-tumorigenic TRAMP C3 cells. Examination of the promoter region of the mouse Nrf2 gene identified a CpG island, which was methylated at specific CpG sites in prostate TRAMP tumor and in TRAMP C1 cells but not in normal prostate or TRAMP C3 cells, as shown by bisulfite genomic sequencing. Reporter assays indicated that methylation of these CpG sites dramatically inhibited the transcriptional activity of the Nrf2 promoter. Chromatin immunopreceipitation (ChIP) assays revealed increased binding of the methyl-CpG-binding protein 2 (MBD2) and trimethyl-histone H3 (Lys9) proteins to these CpG sites in the TRAMP C1 cells as compared to TRAMP C3 cells. In contrast, the binding of RNA Pol II and acetylated histone H3 to the Nrf2 promoter was decreased. Furthermore, treatment of TRAMP C1 cells with DNA methyltransferase (DNMT) inhibitor 5-aza-2′-deoxycytidine (5-aza) and histone deacetylase (HDAC) inhibitor trichostatin A (TSA) restored the expression of Nrf2 as well as the induction of NQO1 in TRAMP C1 cells. Taken together, these results indicate that the expression of Nrf2 is suppressed epigenetically by promoter methylation associated with MBD2 and histone modifications in the prostate tumor of TRAMP mice. Our present findings reveal a novel mechanism by which Nrf2 expression is suppressed in TRAMP prostate tumor, shed new light on the role of Nrf2 in carcinogenesis and provide potential new directions for the detection and prevention of prostate cancer

Author Correction: Cross-ancestry genome-wide association analysis of corneal thickness strengthens link between complex and Mendelian eye diseases.

Emmanuelle Souzeau, who contributed to analysis of data, was inadvertently omitted from the author list in the originally published version of this Article. This has now been corrected in both the PDF and HTML versions of the Article

Association of Genetic Variants With Primary Open-Angle Glaucoma Among Individuals With African Ancestry.

Importance:Primary open-angle glaucoma presents with increased prevalence and a higher degree of clinical severity in populations of African ancestry compared with European or Asian ancestry. Despite this, individuals of African ancestry remain understudied in genomic research for blinding disorders. Objectives:To perform a genome-wide association study (GWAS) of African ancestry populations and evaluate potential mechanisms of pathogenesis for loci associated with primary open-angle glaucoma. Design, Settings, and Participants:A 2-stage GWAS with a discovery data set of 2320 individuals with primary open-angle glaucoma and 2121 control individuals without primary open-angle glaucoma. The validation stage included an additional 6937 affected individuals and 14 917 unaffected individuals using multicenter clinic- and population-based participant recruitment approaches. Study participants were recruited from Ghana, Nigeria, South Africa, the United States, Tanzania, Britain, Cameroon, Saudi Arabia, Brazil, the Democratic Republic of the Congo, Morocco, Peru, and Mali from 2003 to 2018. Individuals with primary open-angle glaucoma had open iridocorneal angles and displayed glaucomatous optic neuropathy with visual field defects. Elevated intraocular pressure was not included in the case definition. Control individuals had no elevated intraocular pressure and no signs of glaucoma. Exposures:Genetic variants associated with primary open-angle glaucoma. Main Outcomes and Measures:Presence of primary open-angle glaucoma. Genome-wide significance was defined as P &lt; 5 × 10-8 in the discovery stage and in the meta-analysis of combined discovery and validation data. Results:A total of 2320 individuals with primary open-angle glaucoma (mean [interquartile range] age, 64.6 [56-74] years; 1055 [45.5%] women) and 2121 individuals without primary open-angle glaucoma (mean [interquartile range] age, 63.4 [55-71] years; 1025 [48.3%] women) were included in the discovery GWAS. The GWAS discovery meta-analysis demonstrated association of variants at amyloid-β A4 precursor protein-binding family B member 2 (APBB2; chromosome 4, rs59892895T&gt;C) with primary open-angle glaucoma (odds ratio [OR], 1.32 [95% CI, 1.20-1.46]; P = 2 × 10-8). The association was validated in an analysis of an additional 6937 affected individuals and 14 917 unaffected individuals (OR, 1.15 [95% CI, 1.09-1.21]; P &lt; .001). Each copy of the rs59892895*C risk allele was associated with increased risk of primary open-angle glaucoma when all data were included in a meta-analysis (OR, 1.19 [95% CI, 1.14-1.25]; P = 4 × 10-13). The rs59892895*C risk allele was present at appreciable frequency only in African ancestry populations. In contrast, the rs59892895*C risk allele had a frequency of less than 0.1% in individuals of European or Asian ancestry. Conclusions and Relevance:In this genome-wide association study, variants at the APBB2 locus demonstrated differential association with primary open-angle glaucoma by ancestry. If validated in additional populations this finding may have implications for risk assessment and therapeutic strategies

Impact of measurement error on testing genetic association with quantitative traits

10.1371/journal.pone.0087044PLoS ONE91-POLN

Genetic Variants on Chromosome 1q41 Influence Ocular Axial Length and High Myopia

As one of the leading causes of visual impairment and blindness, myopia poses a significant public health burden in Asia. The primary determinant of myopia is an elongated ocular axial length (AL). Here we report a meta-analysis of three genome-wide association studies on AL conducted in 1,860 Chinese adults, 929 Chinese children, and 2,155 Malay adults. We identified a genetic locus on chromosome 1q41 harboring the zinc-finger 11B pseudogene ZC3H11B showing genome-wide significant association with AL variation (rs4373767, β = −0.16 mm per minor allele, Pmeta = 2.69×10−10). The minor C allele of rs4373767 was also observed to significantly associate with decreased susceptibility to high myopia (per-allele odds ratio (OR) = 0.75, 95% CI: 0.68–0.84, Pmeta = 4.38×10−7) in 1,118 highly myopic cases and 5,433 controls. ZC3H11B and two neighboring genes SLC30A10 and LYPLAL1 were expressed in the human neural retina, retinal pigment epithelium, and sclera. In an experimental myopia mouse model, we observed significant alterations to gene and protein expression in the retina and sclera of the unilateral induced myopic eyes for the murine genes ZC3H11A, SLC30A10, and LYPLAL1. This supports the likely role of genetic variants at chromosome 1q41 in influencing AL variation and high myopia