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Supplementary Data from High-Resolution Array-Based Comparative Genomic Hybridization of Bladder Cancers Identifies Mouse Double Minute 4 (MDM4) as an Amplification Target Exclusive of MDM2 and TP53

Purpose: Loss of p53 function in urothelial cell carcinoma (UCC) by mutation or inactivation disrupts normal cell cycle checkpoints, generating a favorable milieu for genomic instability, a hallmark of UCC. The aim of this study was to characterize novel DNA copy number changes to identify putative therapeutic targets.Experimental Design: We report our findings using array comparative genomic hybridization on a whole-genome BAC/PAC/cosmid array with a median clone interval of 0.97 Mb to study a series of UCC cases. TP53 status was determined by direct sequencing, and an in-house tissue microarray was constructed to identify protein expression of target genes.Results: Array comparative genomic hybridization allowed identification of novel regions of copy number changes in addition to those already known from previous studies. A novel amplification previously unreported in UCC was identified at 1q32. A chromosome 1 tile path array was used to analyze tumors that showed gains and amplification; the mouse double minute 4 (MDM4) homologue was identified as the amplified gene. MDM4 mRNA expression correlated with copy number and tumor grade. Copy number changes of MDM4 and MDM2 occurred exclusively in tumors with wild-type p53. Overexpression of MDM4 corresponded to disruption of p53 transcriptional activity. Immunohistochemistry on an independent series by tissue microarray identified an inverse relationship between Mdm4 and Mdm2, with Mdm4 expression highest in invasive UCC.Conclusion: The data indicate that gain/amplification and overexpression of MDM4 is a novel molecular mechanism by which a subset of UCC escapes p53-dependent growth control, thus providing new avenues for therapeutic intervention.

BackgroundAmong the most prevalent cancers in the urinary tract is bladder cancer, a caner with a high rate of recurrence and metastasis as compared to other malignancies. To date, there have been many genes reported as potential bladder cancer biomarkers among which is the EN2 gene, a member of the homeobox family containing transcriptional factors. Several studies suggested the overexpression of EN2 to be involved with the development of a number of tumors such as bladder cancer. However, the process of involvement of EN2 in the bladder tumorigenesis remains elusive.MethodsRT-qPCR was carried out to determine the gene expression of 17 cell lines. The short-term silencing of EN2 expression was then implemented in high-expressing cell lines using siRNAs. Using the scratch assay, the outcome of modulating the in vitro EN2 expression on the bladder cancer migration was determined. Correlation between the IC50 values with the EN2 expression was analyzed by correlating the viability of cells following the Newcastle Disease Virus infection with the fold change. Immunohistochemistry was then performed to determine the expression of the EN2 protein in the bladder cancer tissues.ResultsIn the current study, EN2 was differentially expressed in bladder cancer in vitro and upon modulating the expression of EN2, we found a reduction in the migratory effect of bladder cancer in vitro. In addition, following 24 hours post infection, a moderate correlation between EN2 gene expression and NDV-mediated oncolysis was observed. No expression of EN2 in bladder cancer tissues suggesting the need for further studies to investigate the expression of EN2 protein in bladder cancer.ConclusionEN2 may be a potential prognostic or diagnostic bladder cancer biomarker, however, further investigations are required to evaluate the EN2 gene as a potential bladder cancer biomarker.

Bladder cancer cells can acquire persistent infection of oncolytic Newcastle disease virus (NDV) but the molecular mechanism(s) remain unelucidated. This poses a major barrier to the effective clinical translation of oncolytic NDV virotherapy of cancers. To improve our understanding of the molecular mechanism(s) associated with the development of NDV persistent infection in bladder cancer, we used mRNA expression profiles of persistently infected bladder cancer cells to construct PPI networks. Based on paths and modules in the PPI network, the bridges were found mainly in the upregulated mRNA-pathways of p53 signalling, ECM-receptor interaction, and TGF-beta signalling and downregulated mRNA-pathways of antigen processing and presentation, protein processing in endoplasmic reticulum, completement and coagulation cascades in persistent TCCSUPPi cells. In persistent EJ28Pi cells, connections were identified mainly through upregulated mRNA-pathways of renal carcinoma, viral carcinogenesis, Ras signalling and cell cycle and the downregulated mRNA-pathways of Wnt signalling, HTLV-I infection and pathways in cancers. These connections were mainly dependent on RPL8-HSPA1A/HSPA4 in TCCSUPPi cells and EP300, PTPN11, RAC1—TP53, SP1, CCND1 and XPO1 in EJ28Pi cells. Oncomine validation showed that the top hub genes identified in the networks that include RPL8, THBS1, F2 from TCCSUPPi and TP53 and RAC1 from EJ28Pi are involved in the development and progression of bladder cancer. Protein-drug interaction networks identified several putative drug targets that could be used to disrupt the linkages between the modules and prevent bladder cancer cells from acquiring NDV persistent infection. This novel PPI network analysis of differentially expressed mRNAs of NDV persistently infected bladder cancer cell lines provide an insight into the molecular mechanisms of NDV persistency of infection in bladder cancers and the future screening of drugs that can be used together with NDV to enhance its oncolytic efficacy.

Background The Philadelphia (Ph)-negative myeloproliferative neoplasms (MPNs), namely essential thrombocythaemia (ET), polycythaemia vera (PV) and primary myelofibrosis (PMF), are a group of chronic clonal haematopoietic disorders that have the propensity to advance into bone marrow failure or acute myeloid leukaemia; often resulting in fatality. Although driver mutations have been identified in these MPNs, subtype-specific markers of the disease have yet to be discovered. Next-generation sequencing (NGS) technology can potentially improve the clinical management of MPNs by allowing for the simultaneous screening of many disease-associated genes. Methods The performance of a custom, in-house designed 22-gene NGS panel was technically validated using reference standards across two independent replicate runs. The panel was subsequently used to screen a total of 10 clinical MPN samples (ET n = 3, PV n = 3, PMF n = 4). The resulting NGS data was then analysed via a bioinformatics pipeline. Results The custom NGS panel had a detection limit of 1% variant allele frequency (VAF). A total of 20 unique variants with VAFs above 5% (4 of which were putatively novel variants with potential biological significance) and one pathogenic variant with a VAF of between 1 and 5% were identified across all of the clinical MPN samples. All single nucleotide variants with VAFs ≥ 15% were confirmed via Sanger sequencing. Conclusions The high fidelity of the NGS analysis and the identification of known and novel variants in this study cohort support its potential clinical utility in the management of MPNs. However, further optimisation is needed to avoid false negatives in regions with low sequencing coverage, especially for the detection of driver mutations in MPL.

Introduction Lin-11, Isl-1 and Mec-3 domains (LIM) homeobox genes are among the most important sub-families of homeobox genes. These genes are thought to play an important role in cancer. In this study, the protein expression of these genes was examined in urothelial carcinoma of the bladder. The expression pattern of Islet-1 (ISL1) and LIM homeobox 5 (LHX5) across different cancer stages and grades, as well as the association between the protein expression of these genes and patient demographics and clinicopathological features, were examined. Methods A total of 100 formalin-fixed paraffin-embedded urothelial carcinoma tissues were selected from the Department of Pathology, Hospital Kuala Lumpur and the protein expression of ISL1 and LHX5 was determined using immunohistochemistry. Results Positive expression of ISL1 and LHX5 was detected in 94% and 98% of the samples, respectively. There were no distinct LHX5 expression patterns associated with different cancer stages, but the proportion of high-expressing tumours was higher in high-grade tumours. In addition, there was a significant association between the expression of LHX5 and tumour grade. The proportion of tumours expressing high levels of ISL1 was found to be highest in later stage tumours. Conclusion The high percentage of tumours expressing both these genes suggests that ISL1 and LHX5 play an important role in bladder tumourigenesis across multiple stages.

Background Newcastle disease virus (NDV) has been identified as an attractive virotherapeutic agent that targets various type of human cancers while leaving normal cells unharmed. Wild-type NDV strain AF2240 has been found to persistently infect a subpopulation of cancer cells in vitro, making the cells less susceptible to NDV-mediated oncolysis. It is proposed that transcriptome profiling of NDV persistently infected bladder cancer cell lines will provide insights to understand such occurrence by identifying specific pathways associated with NDV persistent infection due to transcriptomic dysregulation. Results Transcriptome profiling revealed a total of 63 and 134 differentially expressed genes (DEGs) from NDV persistently infected TCCSUPPi and EJ28Pi bladder cancer cells relative to their uninfected controls, respectively. Of the 63 DEGs identified for TCCSUPPi cells, 25 DEGs were upregulated (log2 fold-change ≥ 0) and 38 DEGs were downregulated (log2 fold-change ≤ 0). These genes were significantly enriched in the molecular function of calcium binding (GO:0005509) and DNA-binding transcription repressor activity, RNA polymerase II-specific (GO:0001227) and the enriched important upregulated pathways were mainly heme metabolism, TGF-beta signaling and spermatogenesis. As for EJ28Pi, 55 DEGs were upregulated (log2 fold-change ≥ 0) and 79 DEGs were downregulated (log2 fold-change ≤ 0). These DEGs resulted in significantly enriched molecular function such as protein domain specific binding (GO:0019904) and RNA polymerase II regulatory region sequence-specific DNA binding (GO:0000977). The enriched important upregulated pathways were allograft rejection, KRAS signaling up and interferon gamma response. Other important pathways that were downregulated in both the NDV-persistently infected cell lines were angiogenesis, apoptosis, and xenobiotic metabolism. Conclusion The transcriptome profiles (RNA-Seq) of these cell lines suggest that evasion of apoptosis and increase in TGF-beta signaling and interferon gamma response activities are crucial for establishment of NDV persistent infection in bladder cancer cells. Findings from this study provide the molecular basis that warrant further study on how bladder cancer cells acquired NDV persistent infection. Resolving the mechanism of persistent infection will facilitate the application of NDV for more effective treatment of bladder cancer.

MotivationBladder cancer cells acquire persistent infection associated with oncolytic Newcastle disease virus (NDV) in which its molecular events are still unclear. This poses a potential problem for oncolytic virus application for cancer therapy. To unravel the molecular mechanism underlying the development of NDV persistent infection in bladder cancer, we used mRNA expression profile of the persistently infected bladder cancer cells to construct PPI network.ResultsBased on path and module exploring in the PPI network, the bridges were found mainly from pathways of p53 signalling, ECM-receptor interaction, and TGF-beta signalling by the upregulated mRNAs, to the antigen processing and presentation, protein processing in endoplasmic reticulum, completement and coagulation cascades by the downregulated mRNAs in NDV persistent TCCSUPPi cells. In persistent EJ28Pi cells comparatively, connections were identified mainly from pathways of renal carcinoma, viral carcinogenesis, Ras signalling and cell cycle by the upregulated mRNAs, to the Wnt signalling, HTLV-I infection and pathways in cancer by the downregulated mRNAs. This connection was mainly dependent on of RPL8- HSPA1A/HSPA4 in TCCSUPPi cells and EP300, PTPN11, RAC1 - TP53, SP1, CCND1 and XPO1 in EJ28Pi cells. Oncomine validation showed that the top hub genes identified in the network that includes RPL8, THBS1, F2 from TCCSUPPi and TP53 and RAC1 from EJ28Pi are involved in the development and progression of bladder cancer. Protein-drug interaction network, have identified several drugs targets that could be used to disconnect the linkages between modules and prevent bladder cancer cells from acquiring NDV persistent infection. This is the first time reporting the PPI network analysis of differentially expressed mRNAs of the NDV persistently infected bladder cancer cell lines which provide an insight into screening drugs that could be used together with NDV to manage bladder cancer resistance to therapy and progression.

BackgroundNewcastle disease virus (NDV) has been an attractive virotherapy agent that targets various type of human cancers while leaving normal cells unharmed. Wild-type NDV strain AF2240 has been found to persistently infect subpopulation of cancer cellsin vitro, making the cells less susceptible to NDV-mediated oncolysis. It is proposed that transcriptome profiling of NDV persistently infected bladder cancer cell lines will provide insights to understand such occurrence by identifying specific pathways associated with NDV persistent infection due to transcriptomic dysregulation.ResultsTranscriptome profiling revealed a total of 63 and 134 differentially expressed genes (DEGs) from NDV persistently infected TCCSUPPi and EJ28Pi bladder cancer cells relative to their uninfected controls, respectively. Of the 63 DEGs identified for TCCSUPPi cells, 25 DEGs were upregulated (log2fold-change ≥ 0) and 38 DEGs were downregulated (log2fold-change ≤ 0). These genes were significantly enriched in the molecular function of calcium binding (GO:0005509) and DNA-binding transcription repressor activity, RNA polymerase II-specific (GO:0001227) and the enriched important upregulated pathways were mainly heme metabolism, TGF-beta signaling and spermatogenesis. As for EJ28Pi, 55 DEGs were upregulated (log2fold-change ≥ 0) and 79 DEGs were downregulated (log2fold-change ≤ 0). These DEGs resulted in significantly enriched molecular function such as protein domain specific binding (GO:0019904) and RNA polymerase II regulatory region sequence-specific DNA binding (GO:0000977). The enriched important upregulated pathways were allograft rejection, KRAS signaling up and interferon gamma response. Other important pathways that were downregulated in both the NDV-persistently infected cell lines were angiogenesis, apoptosis, and xenobiotic metabolism.ConclusionThe transcriptome profiles (RNA-Seq) of these cell lines suggest that evasion of apoptosis and increase in TGF-beta signaling and interferon gamma response activities are crucial for establishment of NDV persistent infection in bladder cancer cells. Findings from this study provide the molecular basis that warrant further study on how bladder cancer cells acquired NDV persistent infection. Resolving the mechanism of persistent infection will facilitate the application of NDV for more effective treatment of bladder cancer.

Osteomodulin (OMD) and proline/arginine-rich end leucine repeat protein (PRELP) are secreted extracellular matrix proteins belonging to the small leucine-rich proteoglycans family. We found that OMD and PRELP were specifically expressed in umbrella cells in bladder epithelia, and their expression levels were dramatically downregulated in all bladder cancers from very early stages and various epithelial cancers. Our in vitro studies including gene expression profiling using bladder cancer cell lines revealed that OMD or PRELP application suppressed the cancer progression by inhibiting TGF-&beta, and EGF pathways, which reversed epithelial&ndash, mesenchymal transition (EMT), activated cell&ndash, cell adhesion, and inhibited various oncogenic pathways. Furthermore, the overexpression of OMD in bladder cancer cells strongly inhibited the anchorage-independent growth and tumorigenicity in mouse xenograft studies. On the other hand, we found that in the bladder epithelia, the knockout mice of OMD and/or PRELP gene caused partial EMT and a loss of tight junctions of the umbrella cells and resulted in formation of a bladder carcinoma in situ-like structure by spontaneous breakdowns of the umbrella cell layer. Furthermore, the ontological analysis of the expression profiling of an OMD knockout mouse bladder demonstrated very high similarity with those obtained from human bladder cancers. Our data indicate that OMD and PRELP are endogenous inhibitors of cancer initiation and progression by controlling EMT. OMD and/or PRELP may have potential for the treatment of bladder cancer.

Background Newcastle disease virus (NDV) is an oncolytic virus with excellent selectivity against cancer cells, both in vitro and in vivo. Unfortunately, prolonged in vitro NDV infection results in the development of persistent infection in the cancer cells which are then able to resist NDV-mediated oncolysis. However, the mechanism of persistency of infection remains poorly understood. Methods In this study, we established persistently NDV-infected EJ28 bladder cancer cells, designated as EJ28P. Global transcriptomic analysis was subsequently carried out by microarray analysis. Differentially expressed genes (DEGs) between EJ28 and EJ28P cells identified by the edgeR program were further analysed by Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis (IPA) analyses. In addition, the microarray data were validated by RT-qPCR. Results Persistently NDV-infected EJ28 bladder cancer cells were successfully established and confirmed by flow cytometry. Microarray analysis identified a total of 368 genes as differentially expressed in EJ28P cells when compared to the non-infected EJ28 cells. GSEA revealed that the Wnt/β-catenin and KRAS signalling pathways were upregulated while the TGF-β signalling pathway was downregulated. Findings from this study suggest that the upregulation of genes that are associated with cell growth, pro-survival, and anti-apoptosis may explain the survivability of EJ28P cells and the development of persistent infection of NDV. Conclusions This study provides insights into the transcriptomic changes that occur and the specific signalling pathways that are potentially involved in the development and maintenance of NDV persistency of infection in bladder cancer cells. These findings warrant further investigation and is crucial towards the development of effective NDV oncolytic therapy against cancer.

Three tridentate Schiff bases containing N and S donor atoms were synthesized via the condensation reaction between S-2-methylbenzyldithiocarbazate with 2-acetyl-4-methylpyridine (S2APH); 4-methyl-3-thiosemicarbazide with 2-acetylpyridine (MT2APH) and 4-ethyl-3-thiosemicarbazide with 2-acetylpyridine (ET2APH). Three new, binuclear and mixed-ligand copper(II) complexes with the general formula, [Cu(sac)(L)]2 (sac = saccharinate anion; L = anion of the Schiff base) were then synthesized, and subsequently characterized by IR and UV/Vis spectroscopy as well as by molar conductivity and magnetic susceptibility measurements. The Schiff bases were also spectroscopically characterized using NMR and MS to further confirm their structures. The spectroscopic data indicated that the Schiff bases behaved as a tridentate NNS donor ligands coordinating via the pyridyl-nitrogen, azomethine-nitrogen and thiolate-sulphur atoms. Magnetic data indicated a square pyramidal environment for the complexes and the conductivity values showed that the complexes were essentially non-electrolytes in DMSO. The X-ray crystallographic analysis of one complex, [Cu(sac)(S2AP)]2 showed that the Cu(II) atom was coordinated to the thiolate-S, azomethine-N and pyridyl-N donors of the S2AP Schiff base and to the saccharinate-N from one anion, as well as to the carbonyl-O atom from a symmetry related saccharinate anion yielding a centrosymmetric binuclear complex with a penta-coordinate, square pyramidal geometry. All the copper(II) saccharinate complexes were found to display strong cytotoxic activity against the MCF-7 and MDA-MB-231 human breast cancer cell lines.

The ceramide synthase 2 (CERS2) gene has been linked to tumour recurrence and invasion in many different types of cancers including bladder cancer. In this study, the expression levels of CERS2 in bladder cancer cell lines were analysed using qRT-PCR and the protein expression in clinical bladder cancer histopathological specimens were examined via immunohistochemistry. The potential utility of CERS2 as a predictive biomarker of response to oncolytic virotherapy was assessed by correlating the CERS2 mRNA expression to IC50 values of cells treated with the Newcastle disease virus (NDV), AF2240 strain. This study demonstrates that CERS2 is differentially expressed in different types of bladder cancer cell lines and that the siRNA-mediated downregulation of the expression of CERS2 reduces the migratory potential of UMUC1 bladder cancer cells. However, there were no significant correlations between the expression levels of the CERS2 protein with bladder cancer grade/stage or between the IC50 values of cells treated with NDV and CERS2 expression. Although the utility of CERS2 expression may be limited, its potential as an antimigration cancer therapeutic should be further examined.

Background aims Mesenchymal stromal cells (MSCs) have been identified as pan-immunosuppressant in various in vitro and in vivo inflammatory models. Although the immunosuppressive activity of MSCs has been explored in various contexts, the precise molecular signaling pathways that govern inhibitory functions remain poorly elucidated. Methods By using a microarray-based global gene expression profiling system, this study aimed to decipher the underlying molecular pathways that may mediate the immunosuppressive activity of umbilical cord–derived MSCs (UC-MSCs) on activated T cells. Results In the presence of UC-MSCs, the proliferation of activated T cells was suppressed in a dose-depended manner by cell-to-cell contact mode via an active cell-cycle arrest at the G 0 /G 1 phase of the cell cycle. The microarray analysis revealed that particularly, IFNG , CXCL9 , IL2 , IL2RA and CCND3 genes were down-regulated, whereas IL11 , VSIG4 , GFA1 , TIMP3 and BBC3 genes were up-regulated by UC-MSCs. The dysregulated gene clusters associated with immune-response-related ontologies, namely, lymphocyte proliferation or activation, apoptosis and cell cycle, were further analyzed. Conclusions Among the nine canonical pathways identified, three pathways (namely T-helper cell differentiation, cyclins and cell cycle regulation, and gap/tight junction signalling pathways) were highly enriched with these dysregulated genes. The pathways represent putative molecular pathways through which UC-MSCs elicit immunosuppressive activity toward activated T cells. This study provides a global snapshot of gene networks and pathways that contribute to the ability of UC-MSCs to suppress activated T cells.

Twelve tin(IV) compounds (5–16) derived from four tridentate thiosemicarbazone Schiff bases of 4-methyl-3-thiosemicarbazide with 2-hydroxy-3-methoxybenzaldehyde (1, 2) and 4-phenyl-3-thiosemicarbazide with 2,3-dihydroxybenzaldehyde (3, 4) of the general formulae [R2Sn(Ln)] and [Sn(Ln)2] (where R = Ph or Me; Ln = 1, 2, 3 and 4) were synthesized and characterized by elemental analysis, IR, UV–vis, mass spectrometry and multinuclear NMR (1H, 13C and 119Sn) spectroscopy. X-ray crystallographic data was obtained for 11′, a 2:1 co-crystal between Ph2Sn(L2) (11) and 3-methoxysalicylaldehyde azine, and Me2Sn(L2) (12) where L2H2 is 2-(2-hydroxy-3-methoxybenzylidene)-N-phenylhydrazinecarbothioamide. The analysis revealed distinct coordination geometries for 11 and 12 approaching trigonal–bipyramidal. In the crystal of 11′, supramolecular dimers arising from amine N H…S(thiolate) hydrogen bonding and {…HNCS}2 synthons are evident; π(chelate ring)…π(oxidobenzylidene) stacking is also apparent. In the crystal of 12, supramolecular, helical chains are generated by a combination of amine N H…O(phenoxide) hydrogen bonding and Sn…S secondary bonding. The cytotoxic activity of the compounds against a panel of ten cancer cell lines, [HT29 (colon), U87 and SJ-G2 (glioblastoma), MCF-7 (breast), A2780 (ovarian), H460 (lung), A431 (skin), DU145 (prostate), BE2-C (neuroblastoma) and MIA (pancreas), and one normal cell line, MCF-10A (normal breast)] were investigated. The thiosemicarbazone Schiff bases 1 and 4 as well as the diphenyltin(IV) compounds showed a strong ability to inhibit the growth of cancer cells, with particular selectivity against HT29, MCF-7, A2780, A431, BE2-C, SJ-G2 and MIA cell lines. The structure–activity relationship of all these compounds were studied by evaluating the effect of alkyl and aryl groups attached on the thiosemicarbazone backbone, the methoxy/hydroxyl groups present at the meta-position of the phenyl ring and alkyl or aryl groups bound to the tin center.

Six new organotin(IV) compounds of Schiff bases derived from S-R-dithiocarbazate [R = benzyl (B), 2- or 4-methylbenzyl (2M and 4M, respectively)] condensed with 2-hydroxy-3-methoxybenzaldehyde (oVa) were synthesised and characterised by elemental analysis, various spectroscopic techniques including infrared, UV-vis, multinuclear (1H, 13C, 119Sn) NMR and mass spectrometry, and single crystal X-ray diffraction. The organotin(IV) compounds were synthesised from the reaction of Ph2SnCl2 or Me2SnCl2 with the Schiff bases (S2MoVaH/S4MoVaH/SBoVaH) to form a total of six new organotin(IV) compounds that had a general formula of [R2Sn(L)] (where L = Schiff base, R = Ph or Me). The molecular geometries of Me2Sn(S2MoVa), Me2Sn(S4MoVa) and Me2Sn(SBoVa) were established by X-ray crystallography and verified using density functional theory calculations. Interestingly, each experimental structure contained two independent but chemically similar molecules in the crystallographic asymmetric unit. The coordination geometry for each molecule was defined by thiolate-sulphur, phenoxide-oxygen and imine-nitrogen atoms derived from a dinegative, tridentate dithiocarbazate ligand with the remaining positions occupied by the methyl-carbon atoms of the organo groups. In each case, the resulting five-coordinate C2NOS geometry was almost exactly intermediate between ideal trigonal-bipyramidal and square-pyramidal geometries. The cytotoxic activities of the Schiff bases and organotin(IV) compounds were investigated against EJ-28 and RT-112 (bladder), HT29 (colon), U87 and SJ-G2 (glioblastoma), MCF-7 (breast) A2780 (ovarian), H460 (lung), A431 (skin), DU145 (prostate), BE2-C (neuroblastoma) and MIA (pancreatic) cancer cell lines and one normal breast cell line (MCF-10A). Diphenyltin(IV) compounds exhibited greater potency than either the Schiff bases or the respective dimethyltin(IV) compounds. Mechanistic studies on the action of these compounds against bladder cancer cells revealed that they induced the production of reactive oxygen species (ROS). The bladder cancer cells were apoptotic after 24 h post-treatment with the diphenyltin(IV) compounds. The interactions of the organotin(IV) compounds with calf thymus DNA (CT-DNA) were experimentally explored using UV-vis absorption spectroscopy. This study revealed that the organotin(IV) compounds have strong DNA binding affinity, verified via molecular docking simulations, which suggests that these organotin(IV) compounds interact with DNA via groove-binding interactions.

Background Brain-derived neurotrophic factor (BDNF) is a neurotrophin found in abundance in brain regions such as the hippocampus, cortex, cerebellum and basal forebrain. It has been associated with the risk of susceptibility to major depressive disorder (MDD). This study aimed to determine the association of three BDNF variants (rs6265, rs1048218 and rs1048220) with Malaysian MDD patients. Methods The correlation of these variants to the plasma BDNF level among Malaysian MDD patients was assessed. A total of 300 cases and 300 matched controls recruited from four public hospitals within the Klang Valley of Selangor State, Malaysia and matched for age, sex and ethnicity were screened for BDNF rs6265, rs1048218 and rs1048220 using high resolution melting (HRM). Findings BDNF rs1048218 and BDNF rs1048220 were monomorphic and were excluded from further analysis. The distribution of the alleles and genotypes for BDNF rs6265 was in Hardy-Weinberg equilibrium for the controls (p = 0.13) but was in Hardy Weinberg disequilibrium for the cases (p = 0.011). Findings from this study indicated that having BDNF rs6265 in the Malaysian population increase the odds of developing MDD by 2.05 folds (95% CI = 1.48–3.65). Plasma from 206 cases and 206 controls were randomly selected to measure the BDNF level using enzyme-linked immunosorbent assay (ELISA). A significant decrease in the plasma BDNF level of the cases as compared to controls (p

Six new tin(IV) compounds derived from tridentate dinegatively charged ONS dithiocarbazate Schiff bases derived from 2-hydroxy-3-methoxybenzaldehyde (H2L1, H2L2 and H2L3) and 2,3-dihydroxybenzaldehyde (H2L4, H2L5 and H2L6) (where H2Ln = di-acids of Schiff base) are reported. The compounds were characterised by elemental analysis, FT-IR and multinuclear NMR (1H, 13C and 119Sn) spectroscopy. The crystal structures of tin(IV) [S-4-methybenzyl-β-N-(2-hydroxy-3-methoxybenzylmethylene)dithiocarbazate] (2) and tin(IV) [S-benzyl-β-N-(2-hydroxy-3-methoxy benzylmethylene)dithiocarbazate] (3) were determined by X-ray single crystal diffraction analysis. X-ray crystallography showed the molecular geometries in homoleptic 2 and 3 to be quite similar in which the dinegative tridentate ligand coordinated the tin atoms via thiolate-S, phenoxide-O and imine-N atoms. The coordination geometries are based on an octahedron with like-atoms mutually trans. The experimental findings were validated by density functional theory (DFT) calculations at the B3LYP/LanL2DZ/6-311G(d,p) level of theory. All the tin(IV) compounds, except the insoluble compound 2 were screened for their in vitro cytotoxicity against a panel ten of cancer cell lines and one normal breast cell line (MCF-10 A) by MTT assay. Interestingly, the cytotoxicity of five tin(IV) compounds against HT29, MCF7 and MIA was higher than the reference drug, cisplatin.

The Newcastle disease virus (NDV) strain AF2240 is an avian avulavirus that has been demonstrated to possess oncolytic activity against cancer cells. However, to illicit a greater anti-cancer immune response, it is believed that the incorporation of immunostimulatory genes such as IL12 into a recombinant NDV backbone will enhance its oncolytic effect. In this study, a newly developed recombinant NDV that expresses IL12 (rAF-IL12) was tested for its safety, stability and cytotoxicity. The stability of rAF-IL12 was maintained when passaged in specific pathogen free (SPF) chicken eggs from passage 1 to passage 10; with an HA titer of 29. Based on the results obtained from the MTT cytotoxic assay, rAF-IL12 was determined to be safe as it only induced cytotoxic effects against normal chicken cell lines and human breast cancer cells while sparing normal cells. Significant tumor growth inhibition (52%) was observed in the rAF-IL12-treated mice. The in vivo safety profile of rAF-IL12 was confirmed through histological observation and viral load titer assay. The concentration and presence of the expressed IL12 was quantified and verified via ELISA assay. In summary, rAF-IL12 was proven to be safe, selectively replicating in chicken and cancer cells and was able to maintain its stability throughout several passages; thus enhancing its potential as an anti-breast cancer vaccine.

Background: The location of the inferior alveolar nerve (IAN) is generally constant in fully grown mandibles. If we know its average distance from the lower border of the mandible, available bone length from the crest of the edentulous ridge can be estimated by physical measurement of the whole length of mandible in that area. This study aimed to measure the superio-inferior distance of the inferior alveolar nerve (SIDIAN) from the base of the mandible in posterior regions on the right and left side based on cone-beam-computed tomography (CBCT) scans and to evaluate gender and ethnicity-related variations in the Malaysian population. Materials and Methods: A total of 100 CBCT-Digital Imaging and Communications in Medicine files of the patients of 3 ethnic populations (Malay, Chinese and Indian) between the ages of 18 and 80 years were selected for the study. The files were imported onto the iCAT software. The measurements of the SIDIAN to the lower border of the mandible in molar regions were done on both sides. The data was analysed using t-test, one-way analysis of variance test, and correlation coefficient test via the SPSS software. Results: Statistically significant positive correlations were identified between the SIDIAN from the lower border of the mandible in the first and second molar regions within the same side as well as between both sides of the mandible (r ≈ 0.8). There were no statistically significant differences between genders. However, there were statistically significant differences on both molar regions and on both sides in all three ethnic groups (P < 0.05). In general, the SIDIAN from the lower border of the mandible was greatest amongst Chinese and smallest amongst Indians. Conclusions: The strong positive correlations on both sides of the mandible indicate the presence of symmetry. Ethnicity-related variations exist in terms of the location of the IAN in the mandible.

Salicylaldehyde-4-methylthiosemicarbazone (H2MTSali) has been prepared via the condensation reaction of 4-methyl-3-thiosemicarbazide and salicylaldehyde. Four new mixed-ligand copper(II) and nickel(II) complexes with a general formula [M(MTSali)L] (M = Cu2+ or Ni2+; L = co-ligand) were synthesized, where L is either imidazole (im) or benzimidazole (bzim). The Schiff base and its mixed-ligand complexes were characterized by IR and UV/Vis spectroscopy, and the complexes by molar conductivity and magnetic susceptibility measurements. The spectroscopic data indicated that the Schiff base behaves as a tridentate ONS donor ligand coordinating via the phenoxide-oxygen, azomethine-nitrogen, and thiolate-sulphur atoms. Magnetic data indicate a square planar environment for the nickel(II) complexes while molar conductance values indicate that the metal complexes are essentially non-electrolytes in DMSO solution. X-ray crystallography shows Cu(MTSali)bzim (1) and Ni(MTSali)bzim (3) to be isostructural, with the metal(II) ions being coordinated by a N2OS donor set that defines an approximate square planar geometry; in both cases, the benzimidazole is splayed with respect to the coordination plane. The copper(II) complexes were active against MDA-MB-231 and MCF-7 breast cancer cell lines, more so than H2MTSali, whereas the nickel(II) complexes were inactive.

Four new mixed-ligand Ni(II) complexes (1–4) containing imidazole (im) or benzimidazole (bz) and tridentate Schiff bases derived from 2,4-dihydroxybenzaldehyde (24D) and 4-methyl-3-thiosemicarbazide (MT24D) or 4-phenyl-3-thiosemicarbazide (PT24D) were synthesised and characterised using elemental and spectral analysis including FTIR, UV–Vis, 1H NMR, 13C NMR and mass spectrometry for Schiff bases, while the complexes were additionally analysed using ICP-OES, molar conductivity, magnetic susceptibility measurements and single crystal X-Ray diffraction (SXRD) analysis. Magnetic susceptibility indicated a square planar geometry for all the metal complexes while molar conductance values showed that the complexes were non-electrolytes in DMSO. The molecular geometries of the neutral complex molecule in [Ni(MT24D)(bz)](bz). CH3OH (2′), that is 2 co-crystallised with a 1,3-benzimidazole molecule as a methanol solvate, and in the cation of [Ni(MT24D)(im)]O2CMe.2H2O (5), a reaction intermediate for 1, were established by X-ray crystallography. Each featured a trans-N2OS coordination geometry defined by phenoxide-O, imine-N and thiolate-S (2′) or thione-S (5) donors as well as the imine-N donors derived from 1,3-benzimidazole (2′) or imidazole (5) molecules. Systematic variations in geometric parameters were correlated with the form of the tridentate ligand, i.e. di-anionic (2′) or mono-anionic (5). In the crystal of 2′, supramolecular chains were sustained by hydrogen bonding and these were connected into a supramolecular layer by π … π stacking interactions occurring between coordinated benzimidazole rings. In the crystal of 5, hydrogen bonding led to a three-dimensional architecture. The Schiff bases and mixed-ligand Ni(II) complexes were tested for their cytotoxic activitY, but all compounds were inactive against the MDA-MB-231 and MCF-7 breast cancer cell lines. Interestingly, the antibacterial analysis of the compounds showed that the PT24D Schiff base, Ni(MT24D)im, Ni(MT24D)bz, and Ni(PT24D)bz complexes had specific and selective activity against Staphylococcus aureus (S. aureus), Bacillus subtilis (B. subtilis), Propionibacterium acne (P. acne) and Enterobacteraerogenes (E. aerogenes). The DNA binding studies of mixed-ligand Ni(II) complexes against calf thymus DNA revealed that slight hypochromism was observed in the absorption spectra suggesting π-π interactions between the aromatic chromophores and the DNA base pairs where 2 had higher Kb values than 1 thus indicating stronger interactions.

Leukemic cells are hard-to-transfect cell lines. Many transfection reagents which can provide high gene transfer efficiency in common adherent cell lines are not effective to transfect established blood cell lines or primary leukemic cells. This study aims to examine a new class of cationic polymer non-viral vector, PEGylated-dextran-spermine (PEG-D-SPM), to determine its ability to transfect the leukemic cells. Here, the optimal conditions of the complex preparation (PEG-D-SPM/plasmid DNA (pDNA)) were examined. Different weight-mixing (w/w) ratios of PEG-D-SPM/pDNA complex were prepared to obtain an ideal mixing ratio to protect encapsulated pDNA from DNase degradation and to determine the optimal transfection efficiency of the complex. Strong complexation between polymer and pDNA in agarose gel electrophoresis and protection of pDNA from DNase were detected at ratios from 25 to 15. Highest gene expression was detected at w/w ratio of 18 in HL60 and K562 cells. However, gene expression from both leukemic cell lines was lower than the control MCF-7 cells. The cytotoxicity of PEG-D-SPM/pDNA complex at the most optimal mixing ratios was tested in HL60 and K562 cells using MTS assay and the results showed that the PEG-D-SPM/pDNA complex had no cytotoxic effect on these cell lines. Spherical shape and nano-nature of PEG-D-SPM/pDNA complex at ratio 18 was observed using transmission electron microscopy. As PEG-D-SPM showed modest transfection efficiency in the leukemic cell lines, we conclude that further work is needed to improve the delivery efficiency of the PEG-D-SPM.

Rare types of cancer are often not effectively treated by approaches such as chemotherapy and radio- therapy, although their side-effects persist. Immunotherapy has been gaining attention worldwide with growing examples of its anticancer activity demonstrated in vivo. This case report describes a 35-year-old male who suffered from advanced epithelioid sarcoma and underwent 18 cycles of chemotherapy without any significant response, who suffered adverse effects that caused lung collapse. A notable response was observed following the administration of autologous immune enhancement therapy (AIET), which involves a process of isolation, activation and expansion of natural killer (NK) and T cells, which were obtained from the patient's own (autologous) peripheral blood. With the present data and the response of the patient to AIET, it may be proposed that AIET is beneficial for patients suffering from advanced epithelioid sarcoma without producing adverse effects.

Background The introduction of effective novel biomarkers of invasion and metastasis is integral for the advancement of breast cancer management. The present study focused on the identification and evaluation of calreticulin (CRT) as a potential biomarker for breast cancer invasion. Methods Two-dimensional gel protein electrophoresis and MALDI-TOF were utilized in the analysis of fresh-frozen invasive intra-ductal carcinoma specimens. Calreticulin-associated expression was analyzed using immunohistochemistry of FFPE non-malignant/malignant breast specimens. A CRT-knockdown model of MCF7 cell line was developed using siRNA and the CRT genotype/phenotype correlations based on migration and trans-well invasion assays were determined. Finally, microarray-based global gene expression profiling was conducted to elucidate the possible calreticulin pro-invasive regulatory pathways. Results Two-dimensional gel protein electrophoresis and MALDI-TOF analysis showed upregulation of calreticulin expression in tumor tissues as compared to the normal adjacent tissues. Meta-analysis of the immunohistochemical results confirmed significantly higher expression of calreticulin (p

Identifying the differences between infants with non-accidental head injuries (NAHI) and accidental head injuries (AHI) may help alert clinicians to recognize markers of abuse. A retrospective review of infants

Membrane type-1 matrix metalloproteinase (MT1-MMP) is a zinc-binding endopeptidase, which plays a crucial role in tumour growth, invasion and metastasis. We have shown previously that MT1-MMP has higher expression levels in the human urothelial cell carcinoma (UCC) tissue. We show here that siRNA against MT1-MMP blocks invasion in UCC cell lines. Invasion is also blocked by broad-spectrum protease and MMP inhibitors including tissue inhibitor of metalloproteinase-1 and -2. Membrane type-1-MMP can also regulate transcription. We have used expression arrays to identify genes that are differentially transcribed when siRNA is used to suppress MT1-MMP expression. Upon MT1-MMP knockdown, Dickkopf-3 (DKK3) expression was highly upregulated. The stability of DKK3 mRNA was unaffected under these conditions, suggesting transcriptional regulation of DKK3 by MT1-MMP. Dickkopf-3 has been previously shown to inhibit invasion. We confirm that the overexpression of DKK3 leads to decreased invasive potential as well as delayed wound healing. We show for the first time that the effects of MT1-MMP on cell invasion are mediated in part through changes in DKK3 gene transcription.

Additional file 4: Table S4. Invasion assay results.

The matrix metalloproteinases (MMPs) are endopeptidases which break down the extracellular matrix and regulate cytokine and growth factor activity. Several MMPs have been implicated in the promotion of invasion and metastasis in a broad range of tumours including urothelial carcinoma. In this study, RNA from 132 normal bladder and urothelial carcinoma specimens was profiled for each of the 24 human MMPs, the four endogenous tissue inhibitors of MMPs (TIMPs) and several key growth factors and their receptors using quantitative real time RT–PCR. Laser capture microdissection (LCM) of RNA from 22 tumour and 11 normal frozen sections was performed allowing accurate RNA extraction from either stromal or epithelial compartments. This study confirms the over expression in bladder tumour tissue of well-documented MMPs and highlights a range of MMPs which have not previously been implicated in the development of urothelial cancer. In summary, MMP-2, MT1-MMP and the previously unreported MMP-28 were very highly expressed in tumour samples while MMPs 1, 7, 9, 11, 15, 19 and 23 were highly expressed. There was a significant positive correlation between transcript expression and tumour grade for MMPs 1, 2, 8, 10, 11, 12, 13, 14, 15 and 28 (P

The aim of this study was to test the utility of AIMP3, an upstream regulator of DNA damage response following genotoxic stress, as a clinical biomarker in muscle-invasive bladder cancer (MIBC). AIMP3 was identified from a meta-analysis of a global gene-expression dataset. AIMP3 protein expression was determined by immunohistochemistry on a customised bladder cancer tissue-microarray (TMA). The mechanism of gene silencing was probed using methylation-specific PCR. The association between AIMP3 expression, Tp53 transactivity and genomic stability was analysed. In vitro AIMP3 translocation to the nucleus in response to ionising radiation was demonstrated using immunofluorescence. Radiosensitisation effects of siRNA-mediated AIMP3-knockdown were measured using colony forming assays. TMAs derived from patients enrolled in BCON, a Phase III multicentre radiotherapy trial in bladder cancer (ISRCTN45938399) were used to evaluate the association between AIMP3 expression and survival. The prognostic value of AIMP3 expression was determined in a TMA derived from patients treated by radical cystectomy. Loss of AIMP3 expression was frequent in MIBC and associated with impaired Tp53 transactivity and genomic instability. AIMP3-knockdown was associated with an increase in radioresistance. Loss of AIMP3 expression was associated with survival in MIBC patients following radiotherapy (HR = 0.53; 95% CI: 0.36 to 0.78, p = 0.002) but was not prognostic in the cystectomy set. In conclusion, AIMP3 expression is lost in a subset of bladder cancers and is significantly predictive of survival following radiotherapy in MIBC patients.

The aim of this study was to test the utility of AIMP3, an upstream regulator of DNA damage response following genotoxic stress, as a clinical biomarker in muscle-invasive bladder cancer (MIBC). AIMP3 was identified from a meta-analysis of a global gene-expression dataset. AIMP3 protein expression was determined by immunohistochemistry on a customised bladder cancer tissue-microarray (TMA). The mechanism of gene silencing was probed using methylation-specific PCR. The association between AIMP3 expression, Tp53 transactivity and genomic stability was analysed. In vitro AIMP3 translocation to the nucleus in response to ionising radiation was demonstrated using immunofluorescence. Radiosensitisation effects of siRNA-mediated AIMP3-knockdown were measured using colony forming assays. TMAs derived from patients enrolled in BCON, a Phase III multicentre radiotherapy trial in bladder cancer (ISRCTN45938399) were used to evaluate the association between AIMP3 expression and survival. The prognostic value of AIMP3 expression was determined in a TMA derived from patients treated by radical cystectomy. Loss of AIMP3 expression was frequent in MIBC and associated with impaired Tp53 transactivity and genomic instability. AIMP3-knockdown was associated with an increase in radioresistance. Loss of AIMP3 expression was associated with survival in MIBC patients following radiotherapy (HR = 0.53; 95% CI: 0.36 to 0.78, p = 0.002) but was not prognostic in the cystectomy set. In conclusion, AIMP3 expression is lost in a subset of bladder cancers and is significantly predictive of survival following radiotherapy in MIBC patients.

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor involved in angiogenesis-mediated progression of acute myeloid leukemia (AML). Studies have reported the role of soluble form of fms-like tyrosine kinase (sFlT-1) delivery as an antitumor agent by inhibiting VEGF. This study investigates the outcome of delivery of a VEGF165 antagonist, soluble vascular endothelial growth factor receptor, namely sFLT-1, mediating lipofectamine 2000 in acute myeloid leukemic cells. A recombinant plasmid expressing sFLT-1 was constructed and transfected into the K562 and HL60 cells using lipofectamine 2000 transfection reagent. sFLT-1 expression/secretion in pVAX-sFLT-1 transfected cells was verified by RT-PCR and western blot. MTS assay was carried out to evaluate the effect of sFLT-1 on human umbilical vein endothelial cells and K562 and HL60 cellsin vitro. Treatment with pVAX-sFLT-1 showed no association between sFLT-1 and proliferation of infected K562 and HL60 cells, while it demonstrated a significant inhibitory impact on the proliferation of HUVECs. The results of the current study imply that the combination of nonviral gene carrier and sFLT-1 possesses the potential to provide efficient tool for the antiangiogenic gene therapy of AML.

Calreticulin (CRT) as a multi-functional endoplasmic reticulum protein is involved in a spectrum of cellular processes which ranges from calcium homeostasis and chaperoning to cell adhesion and finally malignant formation and progression. Previous studies have shown a contributing role for CRT in a range of different cancers. This present review will focus on the possible roles of CRT in the progression of malignant proliferation and the mechanisms involved in its contribution to cancer invasion.

Lentivirus (LV) encoding woodchuck posttranscriptional regulatory element (WPRE) and central polypurine tract (cPPT) driven by CMV promoter have been proven to act synergistically to increase both transduction efficiency and gene expression. However, the inclusion of WPRE and cPPT in a lentiviral construct may pose safety risks when administered to human. A simple lentiviral construct driven by an alternative promoter with proven extended duration of gene expression without the two regulatory elements would be free from the risks. In a non-viral gene delivery context, gene expression driven by human polybiquitin C (UbC) promoter resulted in higher and more persistent expression in mouse as compared to cytomegalovirus (CMV) promoter. In this study, we measured the efficiency and persistency of green fluorescent protein (GFP) reporter gene expression in cells transduced with LV driven by UbC (LV/UbC/GFP) devoid of the WPRE and cPPT in comparison to the established LV construct encoding WPRE and cPPT, driven by CMV promoter (LV/CMV/GFP). However, we found that LV/UbC/GFP was inferior to LV/CMV/GFP in many aspects: (i) the titer of virus produced; (ii) the levels of reporter gene expression when MOI value was standardized; and (iii) the transduction efficiency in different cell types. The duration of reporter gene expression in selected cell lines was also determined. While the GFP expression in cells transduced with LV/CMV/GFP persisted throughout the experimental period of 14 days, expression in cells transduced with LV/UbC/GFP declined by day 2 post-transduction. In summary, the LV driven by the UbC promoter without the WPRE and cPPT does not exhibit enhanced or durable transgene expression.

Invasive urothelial cell carcinoma (UCC) is characterized by increased chromosomal instability and follows an aggressive clinical course in contrast to non-invasive disease. To identify molecular processes that confer and maintain an aggressive malignant phenotype, we used a high-throughput genome-wide approach to interrogate a cohort of high and low clinical risk UCC tumors. Differential expression analyses highlighted cohesive dysregulation of critical genes involved in the G(2)/M checkpoint in aggressive UCC. Hierarchical clustering based on DNA Damage Response (DDR) genes separated tumors according to a pre-defined clinical risk phenotype. Using array-comparative genomic hybridization, we confirmed that the DDR was disrupted in tumors displaying high genomic instability. We identified DNA copy number gains at 20q13.2-q13.3 (AURKA locus) and determined that overexpression of AURKA accompanied dysregulation of DDR genes in high risk tumors. We postulated that DDR-deficient UCC tumors are advantaged by a selective pressure for AURKA associated override of M phase barriers and confirmed this in an independent tissue microarray series. This mechanism that enables cancer cells to maintain an aggressive phenotype forms a rationale for targeting AURKA as a therapeutic strategy in advanced stage UCC.

Purpose: Loss of p53 function in urothelial cell carcinoma (UCC) by mutation or inactivation disrupts normal cell cycle checkpoints, generating a favorable milieu for genomic instability, a hallmark of UCC. The aim of this study was to characterize novel DNA copy number changes to identify putative therapeutic targets.Experimental Design: We report our findings using array comparative genomic hybridization on a whole-genome BAC/PAC/cosmid array with a median clone interval of 0.97 Mb to study a series of UCC cases. TP53 status was determined by direct sequencing, and an in-house tissue microarray was constructed to identify protein expression of target genes.Results: Array comparative genomic hybridization allowed identification of novel regions of copy number changes in addition to those already known from previous studies. A novel amplification previously unreported in UCC was identified at 1q32. A chromosome 1 tile path array was used to analyze tumors that showed gains and amplification; the mouse double minute 4 (MDM4) homologue was identified as the amplified gene. MDM4 mRNA expression correlated with copy number and tumor grade. Copy number changes of MDM4 and MDM2 occurred exclusively in tumors with wild-type p53. Overexpression of MDM4 corresponded to disruption of p53 transcriptional activity. Immunohistochemistry on an independent series by tissue microarray identified an inverse relationship between Mdm4 and Mdm2, with Mdm4 expression highest in invasive UCC.Conclusion: The data indicate that gain/amplification and overexpression of MDM4 is a novel molecular mechanism by which a subset of UCC escapes p53-dependent growth control, thus providing new avenues for therapeutic intervention.