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Previously, we have reported that PARP1 rs1136410 is significantly associated with increased the risk of gallbladder cancer. We aimed to investigate the association of PARP1 rs1805405 and rs3219088 polymorphisms with risk of GBC and also association of the haplotype and combined effect of PARP1 SNPs (rs1805405 G/A, rs3219088 G/T and rs1136410 A/G). We have also investigated the expression profile of PARP1 and its correlation with polymorphisms, clinical parameters and overall survival. PARP1 polymorphisms were genotyped by PCR-RFLP and the expression profile of PARP1 at mRNA level was analyzed by semi-quantitative PCR. Overall survival was analyzed using Kaplan-Meier plot and Cox-regression analysis. Haplotype analysis of the PARP1 polymorphisms revealed that AGG, AAG and GGT haplotypes are significantly associated with decreased risk of GBC, while AAT, AGT, GGG and GAG haplotypes are significantly associated with increased risk of GBC. Patients with T1+T2 and treated with chemotherapy having risk genotypes of rs1805405 have decreased overall survival. Upregulation of PARP1 is significantly associated with longer overall survival in patients with GBC with different clinical parameters. SNPs rs1136410 and rs1805405 genotypes are significantly associated with PARP1 expression. Haplotype analysis suggests that PARP1 may have a potential role in gallbladder carcinogenesis. [ABSTRACT FROM AUTHOR]

Deregulation of DNA repair mechanisms have been frequently demonstrated in the pathology of cancers including gallbladder cancer. We aimed to investigate the association of ERCC4 rs1800067 (G/A) and ERCC5 rs17655 (G/C) with the predisposition in gallbladder cancer and its prognosis. We have also investigated the prognostic and diagnostic values of expression profiles of ERCC4 and ERCC5 in GBC. Polymorphisms of rs1800067 and rs17655 were genotyped by PCR-RFLP. The expression of these genes was analyzed by semi-quantitative PCR. Overall survival was analyzed using Kaplan-Meier plot and cox-regression analysis. Patients with risk group genotypes of rs17655 have shorter overall survival in patients with presence of gallstone, T1+T2 tumor invasion, absence of lymph node involvement and early stages of tumor. Homozygous wild genotype (GG) of rs1800067 and homozygous mutant genotype (CC) of rs17655 together increases two-fold risk of the disease. The variant genotypes (GC/CC) of rs17655 show significantly higher level of ERCC5 expression. Major allele of ERCC4 rs1800067 and minor allele of ERCC5 rs17655 are significantly associated with increased risk of GBC. Upregulation of ERCC4 and ERCC5 is an early event of development of GBC. [ABSTRACT FROM AUTHOR]

Epigenetic alterations have been reported as one of the risk factors of gallbladder cancer. Promoter hypermethylation is associated with high incidence and poor prognosis of GBC. Bcl-2/adenovirus E1B 19 kDa interacting protein 3 is a pro-apoptotic protein member of Bcl-2 family. Present study was aimed to investigate expression profile and promoter methylation status of BNIP3 in GBC and its correlation with clinico-pathological parameters. The expression analysis and methylation status of BNIP3 was performed by semi-quantitative reverse transcription polymerase chain reaction and Methylation-specific polymerase chain reaction respectively in 84 GBC patients and 29 gallstone tissues (used as normal controls). We demonstrate down regulation of BNIP3 in 56% of the GBC samples. BNIP3 promoter is also frequently hypermethylated (69%) in GBC samples. Interestingly, we found that 69% (40/58) of the BNIP3 promoter hypermethylated samples had also reduced expression of BNIP3. Our data demonstrate significant correlation of the mRNA expression and promoter hypermethylation with late stage and nodal metastasis. Hypermethylation of BNIP3 promoter is associated with low overall survival period. Our results suggest that promoter hypermethylation is an early event and can be a frequent mechanism for downregulation of BNIP3 in GBC. [ABSTRACT FROM AUTHOR]

Background Anoxia leads to a robust generation of reactive oxygen species/nitrogen species which can result in mitochondrial dysfunction and associated cell death in the cerebral cortex of neonates. Aim The present study investigated the pharmacological role of tempol in the treatment of rat neonatal cortical mitochondrial dysfunction induced insult progression (day-1 to day-7) and associated neurobehavioral alterations post-anoxia. Methods Rat pups of 30 h age or postnatal day 2 (PND2) were randomly divided into 5 groups ( n = 5 per group): (1) Control; (2) Anoxia; (3) Anoxia + Tempol 75 mg/kg; (4) Anoxia + Tempol 150 mg/kg; and (5) Anoxia + Tempol 300 mg/kg, and subjected to two episode of anoxia (10 min each) at 24 h of time interval in an enclosed chamber supplied with 100% N 2 . Results Tempol significantly decreased nitric oxide ( NO) formation and simultaneously improved superoxide dismutase (SOD) and catalase (CAT) activities. Further, we observed a significantly ( P < 0.05) improvement in mitochondrial respiration, complex enzyme activities, mitochondrial membrane potential (MMP) along with attenuation of transition pore opening (MPT) after treatment with tempol. Furthermore, tempol decreased expression of mitochondrial Bax, cytochrome-C, caspase-9 and caspase-3 while the increase in expression of cytoplasmic Bax, mitochondrial Bcl-2 on day-7 in cortical region indicating regulation of intrinsic pathway of apoptosis. Further, it improved anoxia-induced neurobehavioral outcome (hanging and reflex latencies). Conclusion Biochemical, molecular and behavioral studies suggest the role of tempol in preserving mitochondrial function and associated neurobehavioral outcomes after neonatal anoxia. [ABSTRACT FROM AUTHOR]

Two groups of renal oncocytomas have been cytogenetically defined by the loss of one or both of chromosomes Y and 1 or by structural rearrangement involving 11q12~q13. We report five renal oncocytomas with structural chromosomal rearrangements involving 11q13 with previously unreported partner chromosomes (namely, 1, 6, and 7). For two of the five cases, a t(6;11)(p21;q13) translocation was revealed; the others had t(1;11)(p13;q13), t(7;11)(q11.2;q13), and t(5;11)(q35; q13). Fluorescence in situ hybridization confirmed translocation of CCND1 at 11q13 to partner chromosomes 5, 6, and 7. Overexpression of cyclin D1, the protein product of CCND1, was detected in three of the five cases (60%) by means of immunohistochemical staining of formalin-fixed, paraffin-embedded tumor sections. In three cases for which fresh tissue was available, Southern blot analysis using the MDL-5 probe for the BCL1 breakpoint did not reveal rearrangement of BCL1. In addition, six consecutive renal oncocytomas diagnosed at our institution between 1999 and 2002 whose karyotypes did not show 11q13 translocations were all negative for cyclin D1 overexpression under immunohistochemical analysis. The findings of CCND1 rearrangement with FISH and correlation with cyclin D1 overexpression under immunohistochemical analysis suggest that cyclin D1 alterations play a role in the subset of renal oncocytomas with 11q translocations, although other genes may also be involved. [Copyright &y& Elsevier]

Aim The fundamental events for cancer progression and metastases include loss of cell adhesion, cell proliferation, anchorage-independent cell growth (evading anoikis), cell migration and cell invasion. All these events leading to cancer progression happen in a favorable nurturing tumor microenvironment. This study was designed to explore the anti-tumor activity of staurosporine (a nonspecific protein kinase inhibitor) in the tumor microenvironment of cervical cancer. Main methods The anti-tumor activity of staurosporine was investigated by cell adhesion assay, colony formation assay, apoptosis assay and quantitative real-time polymerase chain reaction (PCR) in cervical cancer cell lines. Key findings The cell adhesion assay showed that staurosporine induces adhesion of cervical cancer cells to the extracellular matrix (ECM) protein fibronectin. The soft agar colony formation assay showed that staurosporine inhibits both the number and size of colony formation in a dose dependent manner and also induces adherent tendency in the cancer cells. Staurosporine also induces prominent apoptosis in single cell suspensions compared to adherent cells. Stroma cell induced transcription of matrix metalloprotease 1 (MMP1) and matrix metalloprotease 2 (MMP2) in cervical cancer cells was inhibited by staurosporine. Significance Our results indicate that staurosporine induces anti-tumor response in the cervical tumor microenvironment by inhibiting the fundamental events for cancer progression and metastases. The present study represents an attractive area for further research and opens up new avenues towards the understanding of cervical cancer therapeutics. [ABSTRACT FROM AUTHOR]

Adverse reports on the exposure of organisms to dichlorvos (DDVP; an organophosphate insecticide) necessitate studies of organismal resistance/tolerance by way of pharmacological or genetic means. In the context of genetic modulation, a mutation in methuselah ( mth; encodes a class II G-protein-coupled receptor (GPCR)) is reported to extend (~35%) the life span of Drosophila melanogaster and enhance their resistance to oxidative stress induced by paraquat exposure (short term, high level). A lack of studies on organismal tolerance of DDVP by genetic modulation prompted us to examine the protective efficacy of mth mutation in exposed Drosophila. Flies were exposed to 1.5 and 15.0 ng/ml DDVP for 12–48 h to examine oxidative stress endpoints and chemical resistance. After prolonged exposure of flies to DDVP, antioxidant enzyme activities, oxidative stress, glutathione content, and locomotor performance were assayed at various days (0, 10, 20, 30, 40, 50) of age. Flies with the mth mutation ( mth 1 ) showed improved chemical resistance and rescued redox impairment after acute DDVP exposure. Exposed mth 1 flies exhibited improved life span along with enhanced antioxidant enzyme activities and rescued oxidative perturbations and locomotor insufficiency up to middle age (~20 days) over similarly exposed w 1118 flies. However, at late (≥30 days) age, these benefits were undermined. Further, similarly exposed mth- knockdown flies showed effects similar to those observed in mth 1 flies. This study provides evidence of tolerance in organisms carrying a mth mutation against prolonged DDVP exposure and further warrants examination of similar class II GPCR signaling facets toward better organismal health. [ABSTRACT FROM AUTHOR]

VEGF secretion into the tumor microenvironment by cancer cells regulates several oncogenic signaling pathways and cancer-regulated angiogenesis. VEGFR receptors are exclusively present on endothelial cells to maintain their biological homeostasis. The acquisition of unique VEGFR2 receptor and VEGFa in cervical cancer (CC) cells reflects VEGFa/VEGFR2 autocrine machinery. Given the critical role of VEGFR2 in endothelial cell proliferation, migration, and angiogenesis, we explored its function in CC epithelial-mesenchymal transition (EMT) and stemness. Here we report that VEGFR2 regulates cancer-induced angiogenesis and EMT-linked stemness in CC cells via AKT/GSK3β/β-catenin and Snail pathway. Receptor tyrosine kinase inhibitor (RTKi) of VEGFR, Pazopanib (PAZ), shows potential anti-VEGFR2 activity and inhibits VEGFa induced metastatic events such as migration, invasion, and anoikis resistance in CC cells. Similarly, PAZ also attenuates cancer-regulated angiogenesis by inhibiting VE-cadherin internalization in endothelial cells followed by inhibition of endothelial cell migration. Selective depletion of VEGFR2 ligand VEGFa in CC cells also attenuates EMT, metastatic events, and inhibition of cancer-induced angiogenesis. In addition, blocking of VEGFR2 signaling in CC cells via PAZ or shRNA alters the formation of cervical tumorspheres (TS) and their successive generation. Collectively, inhibition of functional VEGFa/VEGFR2 autocrine and paracrine axis ceases the cancer-promoting events in cervical cancer cells. Based on the finding in this study, this oncogenic pathways could be used as a potential therapeutic target in a clinical setting with conventional radio-chemotherapy to achieve synergistic killing of CC cells. [ABSTRACT FROM AUTHOR]

Abstract: Background: Amorphous silica nanoparticles (aSNPs) are used for various applications including food industry. However, limited in vivo studies are available on absorption/internalization of ingested aSNPs in the midgut cells of an organism. The study aims to examine cellular uptake of aSNPs (<30nm) in the midgut of Drosophila melanogaster (Oregon R+) owing to similarities between the midgut tissue of this organism and human and subsequently cellular stress response generated by these nanoparticles. Methods: Third instar larvae of D. melanogaster were exposed orally to 1–100μg/mL of aSNPs for 12–36h and oxidative stress (OS), heat shock genes (hsgs), membrane destabilization (Acridine orange/Ethidium Bromide staining), cellular internalization (TEM) and apoptosis endpoints. Results: A significant increase was observed in OS endpoints in the midgut cells of exposed Drosophila in a concentration- and time-dependent manner. Significantly increased expression of hsp70 and hsp22 along with caspases activation, membrane destabilization and mitochondrial membrane potential loss was also observed. TEM analysis showed aSNPs-uptake in the midgut cells of exposed Drosophila via endocytic vesicles and by direct membrane penetration. Conclusion: aSNPs after their internalization in the midgut cells of exposed Drosophila larvae show membrane destabilization along with increased cellular stress and cell death. General significance: Ingested aSNPs show adverse effects on the cells of GI tract of the exposed organism thus their industrial use as a food-additive may raise concern to human health. [Copyright &y& Elsevier]