Virtual screening and multilevel precision-based prioritisation of natural inhibitors targeting the ATPase domain of human DNA topoisomerase II alpha.

Human DNA topoisomerase II alpha (hTopIIα) is a classic chemotherapeutic drug target. The existing hTopIIα poisons cause numerous side effects such as the development of cardiotoxicity, secondary malignancies, and multidrug resistance. The use of catalytic inhibitors targeting the ATP-binding cavity of the enzyme is considered a safer alternative due to the less deleterious mechanism of action. Hence, in this study, we carried out high throughput structure-based virtual screening of the NPASS natural product database against the ATPase domain of hTopIIα and identified the five best ligand hits. This was followed by comprehensive validation through molecular dynamics simulations, binding free energy calculation and ADMET analysis. On stringent multilevel prioritization, we identified promising natural product catalytic inhibitors that showed high binding affinity and stability within the ligand-binding cavity and may serve as ideal hits for anticancer drug development.Communicated by Ramaswamy H. Sarma.

Whole-genome sequencing unravels novel genetic determinants and regulatory pathways associated with triamcinolone acetonide-induced ocular hypertension.

Glucocorticosteroids commonly used to treat certain ocular inflammatory conditions cause an unwarranted elevation in intraocular pressure (IOP) leading to steroid-induced ocular hypertension (OHT). This study aims to identify novel genetic variants in the Indian population associated with steroid responsiveness, specifically to that of intravitreal Triamcinolone acetonide (TA) injections, which leads to OHT in 27% of the TA-treated Indian subjects. Genetic determinants and pathways regulating TA-OHT progression were investigated by applying whole-genome sequencing (WGS) on DNA extracted from 53 blood samples that included TA responders and non-responders. Sequencing analysis yielded 45 intronic and 49 exonic variants to be associated with TA-OHT, which are known to play a vital role in eye, heart, brain, and bone deformities. Of these, the most significant genetic variant associated with TA-OHT was further considered for molecular dynamics (MD) simulation studies. Variants in the CRPPA, PLOD1, ARHGAP1, TIMELESS and TNFSF4 genes were found to be directly implicating TA-OHT. Furthermore, these genes were enriched in pathways associated with cardiomyopathy, focal adhesion, extracellular matrix, and actin cytoskeleton reorganization. MD simulation studies revealed that the top significant variant (rs141625803) in the CRPPA gene possesses a high pathogenic and structurally destabilizing effect. Thus, novel genetic variants that could be significantly associated with the TA-OHT progression were identified in this study. Validation of these targets in a larger cohort of patients along with their functional analysis would inform on the disease, thereby adding to the existing knowledge on the pathophysiology of TA-OHT.

Graphitic Carbon Nitride Causes Widespread Global Molecular Changes in Epithelial and Fibroblast Cells.

For scaffold and imaging applications, nanomaterials such as graphene and its derivatives have been widely used. Graphitic carbon nitride (g-C3N4) is among one such derivative of graphenes, which draws strong consideration due to its physicochemical properties and photocatalytic activity. To use g-C3N4 for biological applications, such as molecular imaging or drug delivery, it must interact with the epithelium, cross the epithelial barrier, and then come in contact with the extracellular matrix of the fibroblast cells. Thus, it becomes essential to understand its molecular mechanism of action. Hence, in this study, to understand the molecular reprogramming associated with g-C3N4, global gene expression using DNA microarrays and proteomics using tandem mass tag (TMT) labeling and mass spectrometry were performed in epithelial and fibroblast cells, respectively. Our results showed that g-C3N4 can cross the epithelial barrier by regulating the adherens junction proteins. Further, using g-C3N4-PDMS scaffolds as a mimic of the extracellular matrix for fibroblast cells, the common signaling pathways were identified between the epithelium and fibroblast cells. These pathways include Wnt signaling, integrin signaling, TGF-ß signaling, cadherin signaling, oxidative stress response, ubiquitin proteasome pathway, and EGF receptor signaling pathways. These altered signature pathways identified could play a prominent role in g-C3N4-mediated cellular interactions in both epithelial and fibroblast cells. Additionally, ß catenin, EGFR, and MAP2K2 protein-protein interaction networks could play a prominent role in fibroblast cell proliferation. The findings could further our knowledge on g-C3N4-mediated alterations in cellular molecular signatures, enabling the potential use of these materials for biological applications such as molecular imaging and drug delivery.

Assessment of single nucleotide polymorphisms associated with steroid-induced ocular hypertension.

AIM: To access the association of forty-eight single nucleotide polymorphisms (SNPs) identified from Caucasian population with steroid-induced ocular hypertension (OHT) in India population. METHODS: Fifty-four triamcinolone-acetonide (TA) and forty-seven dexamethasone (Dex) administered subjects were enrolled in the study after a written consent. Intraocular pressure (IOP) values were recorded for a period of 6-month post steroid injections and patients were grouped as steroid-responders (SR: IOP≥21 mm Hg) and non-responders (NR: IOP≤20 mm Hg). Genomic DNA was isolated from peripheral venous blood. Forty-eight SNPs identified in TA treated Caucasian patients by genome wide association study (GWAS) were genotyped using iPLEX™ MassARRAY among TA as well as Dex administered Indian patients. Genotyping data of 48 general subjects from a previous study were considered as reference controls for statistical analysis. Genotypic frequencies were calculated and P-value, Chi-square and odds ratio at 95% confidence-interval of group A (steroid treated vs controls), group B (SR vs NR), group C (phenotype correlation: influence of time, severity and gender on IOP rise), were calculated. P<0.05 was considered to be statistically significant. RESULTS: OHT was observed in 50% of TA and 26% of Dex administered patients, respectively. IOP rise was mostly severe (>30 mm Hg) and immediate (<1wk) among TA-SR patients while it was noticed to be mild (<30 mm Hg) and between 1-2mo among Dex-SR patients. Logistic regression for risk factor correlation with OHT remained non-significant, hence these factors were not considered as confounding parameters for further analysis. rs133, rs34016742, rs274554, rs10936746, rs274547, rs804854, rs7751500, rs359498, and rs7547448 SNPs significantly varied even after Bonferroni corrections (P<0.0025; group A). rs1879370 (TA) and rs6559662 (Dex) were significantly (P<0.05) associated with OHT (group B). rs133 (severe IOP rise), rs11047639 and rs1879370 (male gender), and rs11171569 (immediate IOP rise) significantly (P<0.05) influenced the phenotype correlation only among TA-OHT patients. However, the significance of these SNPs in group B and phenotype analysis (group C) was lost upon Bonferroni corrections (P<0.0025). CONCLUSION: Prevalence of OHT in study population is observed to be similar to other studies both in TA and Dex treated patients. We can correlate rs34016742 involved in diabetes signaling pathway to the occurrence of ocular edematous and inflammatory conditions. Except rs133 that is involved in neuro-degeneration and myopia occurrence, none of the other SNPs identified in Caucasian population possess any correlation with OHT incidence in TA and Dex administered Indian subjects.

Gene expression profiling of tumor stroma interactions in retinoblastoma.

We aimed to identify the critical molecular pathways altered upon tumor stroma interactions in retinoblastoma (RB). In vitro 2 D cocultures of RB tumor cells (Weri-Rb-1 and NCC-RbC-51) with primary bone marrow stromal cells (BMSC) was established. Global gene expression patterns in coculture samples were assessed using Affymetrix Prime view human gene chip microarray and followed with bioinformatics analyses. Key upregulated genes from Weri-Rb-1 + BMSC and NCC-RbC-51 + BMSC coculture were validated using qRT-PCR to ascertain their role in RB progression. Whole genome microarray experiments identified significant (P ≤ 0.05, 1.1 log 2 FC) transcriptome level changes induced upon coculture of RB cells with BMSC. A total of 1155 genes were downregulated and 1083 upregulated in Weri-Rb-1 + BMSC coculture. Similarly, 1865 genes showed downregulation and 1644 genes were upregulation in NCC-RbC-51 + BMSC coculture. The upregulated genes were significantly associated with pathways of focal adhesion, PI3K-Akt signalling, ECM-receptor interaction, JAK-STAT, TGF-ß signalling thus contributing to RB progression. Validation of key genes by qRT-PCR revealed significant overexpression of IL8, IL6, MYC and SMAD3 in the case of Weri-Rb-1 + BMSC coculture and IL6 in the case of NCC-RbC-51 + BMSC coculture. The microarray expression study on in vitro RB coculture models revealed the pathways that could be involved in the progression of RB. The gene signature obtained in a stimulated model when a growing tumor interacts with its microenvironment may provide new horizons for potential targeted therapy in RB.

Synthesis of saporin-antibody conjugates for targeting EpCAM positive tumour cells.

Epithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein involved in cell proliferation and differentiation. Ribosomal inactivating proteins derived from plants specifically target ribosomes and irreversibly inhibit protein synthesis. EpCAM antibody and saporin were conjugated using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride/N-hydroxysuccinimide chemistry. The mass of the conjugates were characterised using matrix-assisted laser desorption ionisation (MALDI). The saporin-EpCAM (SAP-EpAB) conjugates were tested in-vitro against MCF-7 (breast cancer cells), WERI-Rb1 (retinoblastoma) cells. The flow cytometry and fluorescence microscopy were performed to show the binding efficiency of SAP-EpAB conjugate. Whole transcriptome changes of sap-conjugate treated cells were studied using affymetrix microarrays. MALDI-TOF analysis and polyacrylamide gel electrophoresis confirmed the conjugation of SAP with EpCAM antibody. Flow cytometry and fluorescent microscopy analysis revealed the binding of SAP-EpAB conjugates to the MCF-7, WERI-Rb1 cells. Apoptosis assay by annexin-V has shown an increased apoptotic and necrotic population in conjugate treated cells. MTT assay confirmed the tumour cell death and had shown the IC50 value of 0.8 µg for conjugate in MCF-7 (breast cancer cells), and 1 µg for WERI-Rb1 (retinoblastoma) cells. The microarray analysis revealed downregulation of the tumourigenic genes and upregulation of pro-apoptotic genes leading to apoptosis of tumour cells.

Neocarzinostatin, Aptamer Conjugates for Targeting EpCAM-positive Tumor Cells.

BACKGROUND/AIM: The aim of this study was to investigate the role of Neocarzinostatin (NCS) conjugated with epithelial cell adhesion molecule (EpCAM) aptamer in EpCAM-positive cancer cells. NCS is an antitumor antibiotic protein chromophore that has the ability to cleave double stranded DNA and can be used as a potential drug for the treatment of EpCAM-positive cancers. EpCAM aptamer is an oligonucleotide ligand that binds specifically to EpCAM, a protein overexpressed in tumor cells. MATERIALS AND METHODS: NCS was conjugated with EpCAM aptamer using Sulfo-Succinimidyl 6-(3-(2-pyridyldithio) - propionamide hexanoate) LC-(SPDP) cross-linker to deliver it to EpCAM-positive tumor cells. The conjugates were characterized using polyacrylamide gel electrophoresis (PAGE) and high-performance liquid chromatography (HPLC). Flow cytometry was used to study the binding efficiency of the aptamer and the conjugates in cancer cells. The effect of the conjugate on cancer cells was studied using propidium iodide (PI) to analyze the cell cycle phase changes. The apoptosis assay was performed using the IC50 concentration of NCS. Microarrays were performed to study the gene level changes in cancer cells upon treatment with NCS and the conjugate. RESULTS: Flow cytometry revealed significant binding of aptamer and conjugate in the MCF-7 and WERI-Rb1 cell lines. Briefly, 62% in MCF and 30% in WERI-Rb1 cells with conjugate treated cells (p<0.005). The cell-cycle analysis indicated G2 phase arrest in MCF-7 cells and S phase arrest in WERI-Rb1 cells (p<0.005). Microarray analysis showed differentially expressed genes involved in cell cycle, DNA damage, and apoptosis. The BrDU assay and the apoptosis assay showed that the expression of BrDU was reduced in conjugate-treated cells and the PARP levels were increased confirming the double stranded DNA breaks (p<0.005). In MCF-7 and WERI-Rb1 cells, most of the cells underwent necrosis (p<0.005). CONCLUSION: The EpCAM aptamer conjugated NCS showed specificity to EpCAM-positive cells. The effect of the conjugates on cancer cells were impressive as the conjugate arrested the cell cycle and promoted apoptosis and necrosis. The high levels of PARP expression confirmed the DNA breaks upon conjugate treatment. Our study demonstrates that the NCS conjugated with EpCAM can be targeted to cancer cells sparing normal cells.

Monitoring of changes in lipid profiles during PLK1 knockdown in cancer cells using DESI MS.

The importance of the polo-like kinase 1 (PLK1) gene is increasing substantially both as a biomarker and as a target for highly specific cancer therapy. This is due to its involvement in multiple points of cell progression and carcinogenesis. PLK1 inhibitors' efficacy in treating human cancers has been limited due to the lack of a specific targeting strategy. Here, we describe a method of targeted downregulation of PLK1 in cancer cells and the concomitant rapid detection of surface lipidomic perturbations using desorption electrospray ionization mass spectrometry (DESI MS). The efficient delivery of siRNA targeting PLK1 gene selectively to the cancer cells is achieved by targeting overexpressed cell surface epithelial cell adhesion molecule (EpCAM) by the EpDT3 aptamer. The chimeric aptamer (EpDT3-siPLK1) showed the knockdown of PLK1 gene expression and PLK1 protein levels by quantitative PCR and western blotting, respectively. The abundant surface lipids, phosphatidylcholines (PCs), such as PC(32:1) (m/z 754.6), PC(34:1) (m/z 782.6), and PC(36:2) (m/z 808.6), were highly expressed in MCF-7 and WERI-RB1 cancer cells compared to normal MIO-M1 cells and they were observed using DESI MS. These overexpressed cell surface lipids in the cancer cells were downregulated upon the treatment of EpDT3-siPLK1 chimera indicating a novel role of PLK1 to regulate surface lipid expression in addition to the efficient selective cancer targeting ability. Our results indicate that DESI MS has a potential ability to rapidly monitor aptamer-mediated cancer therapy and accelerate the drug discovery process. Graphical abstract Binding of aptamer chimera to the cells and changes in lipid profile.

Hypermethylation of adenomatosis polyposis coli-2 and its tumor suppressor role in retinoblastoma.

PURPOSE: Retinoblastoma (RB) is a progressive eye cancer of infancy and childhood. Hypermethylation, epigenetic silencing of genes is one of the key events in tumorigenesis. The purpose of this study is to investigate hypermethylation of adenomatosis polyposis coli homologue, APC-2 and possible interaction of APC-2 with Wnt signaling ß-catenin protein in Retinoblastoma. METHODOLOGY: Primary RB tumor samples and cell line were used for the study. DNA isolation, bisulfite conversion, methylation specific PCR and DNA sequencing analysis of PCR products were performed to identify CpG islands and methylation in primary RB tumor samples (n = 30). Chemical demethylation and retrieval of APC-2 expression was studied using 5-Azacytidine (5'-AZC). Flow cytometry, immunofluorescence, western blot were performed for APC-2 expression analysis in demethylated Y79 cells. Co-localization study was conducted to understand the interaction between APC-2 and ß-catenin. RESULTS: APC-2 gene was methylated and down regulated in primary RB tumors. We observed that 70% of RB tumors (21/30) showed positivity with APC-2 methylation. The RB Y79 cells after treatment with demethylating agent 5'-AZC retrieved APC-2 expression, which was confirmed by immunofluorescence and Western blot. Flow cytometry showed APC-2 expression of 29.22% in 5'-AZC treated cells. Co-localization study showed interaction of APC-2 and RB upregulated ß-catenin in Y79 cells. CONCLUSION: We report that APC-2 gene is hypermethylated in both RB tumor samples and Y79 cells. Reduced APC-2 lead to increased Wnt signaling pathway protein, ß-catenin suggesting tumor suppressive role of APC-2 gene.