Arid/semi-arid flora as a treasure trove of bioactives and bioenergy: the case for underutilized desert legumes towards environmental sustainability.

As the spectre of climate change gains in strength with each passing moment, many of our mundane food crops like rice face the heat, leading to uncertain yields and unforeseen disease outbreaks. Subsequently, mankind is forced to look for alternative food choices that should primarily come from indigenous plants that are less demanding in terms of usage of water and application of chemical-based fertilizers/pesticides. There are plants growing in the wild in the arid and semi-arid zones of Rajasthan, India, that can come to the rescue, with an added potential for development into valuable functional foods-i.e., not only as source of carbohydrates, proteins, and micro-nutrients but also that of health benefiting nutraceuticals (like antioxidant flavonoids) and relevant enzymes. The other parts (non-edible) of these plants have often also been traditionally validated via diverse ethnomedicinal practices; these could also be useful bioenergy sources. Keeping in mind the broader aim of looking at future functional foods that are also required to be environmentally sustainable, the current report: (a) reviews the extant literature on underutilized legumes from arid/semi-arid zones, (b) discusses current status with respect to biological activities present therein, and (c) suggests pertinent research questions and solution paths in the domains of bioactives, bioenergy, and sustainable environment.

Metabolic pathway and role of individual species in the bacterial consortium for biodegradation of azo dye: A biocalorimetric investigation.

In this study, an attempt was made to investigate the functional role and metabolic behaviour of the monoculture (Staphylococcus lentus (SL), Bacillus flexus (BF) and Pseudomonas aeruginosa (PA)) in the bacterial biocenosis for biotransformation of an azo dye. The power-time profile obtained from consortia depicted three distinct peaks, which correlated well with the individual bacterial growth (PA > SL > BF), indicating the synergistic relation and division of labour in the biocenosis. The heat release pattern was used to identify the sequential behaviour of microbial consortia in real time. Yield calculation based on total heat liberated to the complete substrate utilization Y (Q/S) for PA, SL, and BF were 15.99, 16.68, 7.32 kJ/L respectively. Similarly, the oxy calorific values Y (Q/O) for the above species are respectively 386, 375, 440 kJ/mol and indicates the aerobic nature of microorganism employed. Further, the metabolome produced during the biotransformation were identified using Gas Chromatography-Mass Spectrometry (GC-MS), based on which a plausible pathway was predicted. The abundant metabolites were palmitic acid (m/z = 256) and diethyl phthalate (m/z = 222.2). The abundance of diethyl phthalate was much lesser in the consortia compared to the monoculture. Thus, the biocalorimetric heat yield calculation along with the stoichiometry and plausible pathway based biochemical elucidation provides a mechanistic basis for understanding the azo-dye biotransformation by the monocultures in consortia.

Comparative docking of dual conformations in human fatty acid synthase thioesterase domain reveals potential binding cavity for virtual screening of ligands.

Human fatty acid synthase (hFASN), a homo dimeric lipogenic enzyme with seven catalytic domains, is an important clinical target in cancer, metabolic syndrome and infections. Here, molecular modelling and docking methods were implemented to examine the inter-molecular interactions of thioesterase (TE) domain in hFASN with its physiological substrate, and to identify potential chemical inhibitors. TE catalyses the hydrolysis of thioester bond between palmitate and the 4' phosphopantetheine of acyl carrier protein, releasing 16-carbon palmitate. The crystal structure of hFASN TE in two inhibitory conformations (A and B) were geometry-optimized and used for molecular docking with palmitate, orlistat (a known FASN inhibitor) and virtual screening against compounds from National Cancer Institute (NCI) database. Relatively, low binding affinity was observed during the complex formation of palmitate with A (-.164 kcal/mol) and B (-.332 kcal/mol) forms of TE, when compared with orlistat-docked TE (A form: -5.872 kcal/mol and B form: -5.484 kcal/mol), clearly indicating that the native inhibited conformation (crystal structure) was unfavourable for substrate binding. We used these orlistat dual binding modes as positive controls for prioritizing the ligands during virtual screening. From 2, 31,617 molecules in the NCI database, 916 high-scoring compounds (hit ligands) were obtained for A-form and 4582 for B-form of the TE-domain, which were then ranked according to glide docking score, XP H bond score, absorption, distribution, metabolism and excretion and binding free energy (Prime/MM-GBSA). Consequently, two top scoring ligands (NSC: 319661 and NSC: 153166) emerged as promising drug candidates that may be tested in FASN-over-expressing diseases.

Targeting HMGA2 in Retinoblastoma Cells in vitro Using the Aptamer Strategy.

High-mobility group A2 (HMGA2) protein regulates retinoblastoma (RB) cancer cell proliferation. Here, a stable phosphorothioate-modified HMGA2 aptamer was used to block HMGA2 protein function in RB cells. HMGA2-aptamer internalisation in RB cells (Y79, Weri Rb1) and non-neoplastic human retinal cells (MIO-M1) were optimised. Aptamer induced dose-dependent cytotoxicity in RB cancer cells (0.25-1.5 µM). Increased expression of TGFß, SMAD4, CDH1, BAX, CASP 3, PARP mRNA and decreased SNAI1, Bcl2 mRNA levels in aptamer-treated RB cells suggests the activation of TGFß-SMAD4-mediated apoptotic pathway. Synergistic effect with etoposide was observed in aptamer treated RB cells (p value ≤0.05). No significant toxicity was observed in non-neoplastic retinal cells.

In Silico Structure Prediction of Human Fatty Acid Synthase-Dehydratase: A Plausible Model for Understanding Active Site Interactions.

Fatty acid synthase (FASN, UniProt ID: P49327) is a multienzyme dimer complex that plays a critical role in lipogenesis. Consequently, this lipogenic enzyme has gained tremendous biomedical importance. The role of FASN and its inhibition is being extensively researched in several clinical conditions, such as cancers, obesity, and diabetes. X-ray crystallographic structures of some of its domains, such as ß-ketoacyl synthase, acetyl transacylase, malonyl transacylase, enoyl reductase, ß-ketoacyl reductase, and thioesterase, (TE) are already reported. Here, we have attempted an in silico elucidation of the uncrystallized dehydratase (DH) catalytic domain of human FASN. This theoretical model for DH domain was predicted using comparative modeling methods. Different stand-alone tools and servers were used to validate and check the reliability of the predicted models, which suggested it to be a highly plausible model. The stereochemical analysis showed 92.0% residues in favorable region of Ramachandran plot. The initial physiological substrate ß-hydroxybutyryl group was docked into active site of DH domain using Glide. The molecular dynamics simulations carried out for 20 ns in apo and holo states indicated the stability and accuracy of the predicted structure in solvated condition. The predicted model provided useful biochemical insights into the substrate-active site binding mechanisms. This model was then used for identifying potential FASN inhibitors using high-throughput virtual screening of the National Cancer Institute database of chemical ligands. The inhibitory efficacy of the top hit ligands was validated by performing molecular dynamics simulation for 20 ns, where in the ligand NSC71039 exhibited good enzyme inhibition characteristics and exhibited dose-dependent anticancer cytotoxicity in retinoblastoma cancer cells in vitro.

Targeting HSP90/Survivin using a cell permeable structure based peptido-mimetic shepherdin in retinoblastoma.

BACKGROUND: Retinoblastoma (RB) is a childhood retinal malignancy. Effective therapeutic strategies are still being investigated in RB disease management. Here, the anti-cancer effect of shepherdin, a peptido-mimetic inhibiting heat shock protein (HSP90)-Survivin interaction has been analyzed. METHODS: We analyzed HSP (HSP70/90) and Survivin protein expressions by immunohistochemistry (29 archival tumors), qRT-PCR, FACS and Western analysis (10 un-fixed RB tumors). We also analyzed cellular cytotoxicity and anti-proliferative effect in peptide treated RB cells (Y79, Weri Rb1) and MIO-M1 cells. RESULTS: Heterogeneous expressions of HSP70/90 and Survivin with a significant association between HSP70 and HSP90 (r(2) = 0.59, p = 0.001) was observed. In RB cells, anti-tumor effects were detected with 0.42 µg/ml of shepherdin at 4 h s of serum starvation. Decreased Survivin, Bcl2, MMP-2 activity with increased Bax, Bim, and Caspase-9 protein expressions were noticed. No significant changes were observed in shepherdin treated non-neoplastic MIO-M1, nor in scramble-peptide treated RB cells. CONCLUSION: The presence of HSPs (HSP70/90) and Survivin reveals multiple cellular mechanisms adopted by RB cells during cancer progression. Serum starvation induced HSP90 whose interactions with Survivin were specifically inhibited by shepherdin. The associated molecular shuffling has been reported. These findings strongly implicate the potential of targeting HSP90-Survivin interaction as an adjuvant therapy in RB management.

Clinico-Pathological Association of Delineated miRNAs in Uveal Melanoma with Monosomy 3/Disomy 3 Chromosomal Aberrations.

PURPOSE: To correlate the differentially expressed miRNAs with clinico-pathological features in uveal melanoma (UM) tumors harbouring chromosomal 3 aberrations among South Asian Indian cohort. METHODS: Based on chromosomal 3 aberration, UM (n = 86) were grouped into monosomy 3 (M3; n = 51) and disomy 3 (D3; n = 35) by chromogenic in-situ hybridisation (CISH). The clinico-pathological features were recorded. miRNA profiling was performed in formalin fixed paraffin embedded (FFPE) UM samples (n = 6) using Agilent, Human miRNA microarray, 8x15KV3 arrays. The association between miRNAs and clinico-pathological features were studied using univariate and multivariate analysis. miRNA-gene targets were predicted using Target-scan and MiRanda database. Significantly dys-regulated miRNAs were validated in FFPE UM (n = 86) and mRNAs were validated in frozen UM (n = 10) by qRT-PCR. Metastasis free-survival and miRNA expressions were analysed by Kaplen-Meier analysis in UM tissues (n = 52). RESULTS: Unsupervised analysis revealed 585 differentially expressed miRNAs while supervised analysis demonstrated 82 miRNAs (FDR; Q = 0.0). Differential expression of 8 miRNAs: miR-214, miR-149*, miR-143, miR-146b, miR-199a, let7b, miR-1238 and miR-134 were studied. Gene target prediction revealed SMAD4, WISP1, HIPK1, HDAC8 and C-KIT as the post-transcriptional regulators of miR-146b, miR-199a, miR-1238 and miR-134. Five miRNAs (miR-214, miR146b, miR-143, miR-199a and miR-134) were found to be differentially expressed in M3/ D3 UM tumors. In UM patients with liver metastasis, miR-149* and miR-134 expressions were strongly correlated. CONCLUSION: UM can be stratified using miRNAs from FFPE sections. miRNAs predicting liver metastasis and survival have been identified. Mechanistic linkage of de-regulated miRNA/mRNA expressions provide new insights on their role in UM progression and aggressiveness.

Global gene deregulations in FASN silenced retinoblastoma cancer cells: molecular and clinico-pathological correlations.

Activation of fatty acid synthase (FASN) enzyme in the de novo lipogenic pathway has been reported in various cancers including retinoblastoma (RB), a pediatric ocular cancer. The present study investigates lipogenesis-dependent survival of RB cancer cells and the associated molecular pathways in FASN silenced RB cells. The siRNA-mediated FASN gene knockdown in RB cancer cells (Y79, WERI RB1) repressed FASN mRNA and protein expressions, and decreased cancer cell viability. Global gene expression microarray analysis was performed in optimized FASN siRNA transfected and untransfected RB cells. Deregulation of various downstream cell signaling pathways such as EGFR (n = 55 genes), TGF-beta (n = 45 genes), cell cycle (n = 41 genes), MAPK (n = 39 genes), lipid metabolism (n = 23 genes), apoptosis (n = 21 genes), GPCR signaling (n = 21 genes), and oxidative phosporylation (n = 18 genes) were observed. The qRT-PCR validation in FASN knockdown RB cells revealed up-regulation of ANXA1, DAPK2, and down-regulation of SKP2, SREBP1c, RXRA, ACACB, FASN, HMGCR, USP2a genes that favored the anti-cancer effect of lipogenic inhibition in RB. The expression of these genes in primary RB tumor tissues were correlated with FASN expression, based on their clinico-pathological features. The differential phosphorylation status of the various PI3K/AKT pathway proteins (by western analysis) indicated that the FASN gene silencing indeed mediated apoptosis in RB cells through the PI3K/AKT pathway. Scratch assay clearly revealed that FASN silencing reduced the invading property of RB cancer cells. Dependence of RB cancer cells on lipid metabolism for survival and progression is implicated. Thus targeting FASN is a promising strategy in RB therapy.

Computational and in vitro Investigation of miRNA-Gene Regulations in Retinoblastoma Pathogenesis: miRNA Mimics Strategy.

PURPOSE: Retinoblastoma (RB), a primary pediatric intraocular tumor, arises from primitive retinal layers. Several novel molecular strategies are being developed for the clinical management of RB. miRNAs are known to regulate cancer-relevant biological processes. Here, the role of selected miRNAs, namely, miR-532-5p and miR-486-3p, has been analyzed for potential therapeutic targeting in RB. METHODS: A comprehensive bioinformatic analysis was performed to predict the posttranscriptional regulators (miRNAs) of the select panel of genes [Group 1: oncogenes (HMGA2, MYCN, SYK, FASN); Group 2: cancer stem cell markers (TACSTD, ABCG2, CD133, CD44, CD24) and Group 3: cell cycle regulatory proteins (p53, MDM2)] using Microcosm, DIANALAB, miRBase v 18, and REFSEQ database, and RNA hybrid. The expressions of five miRNAs, namely, miR-146b-5p, miR-532-5p, miR-142-5p, miR-328, and miR-486-3p, were analyzed by qRT-PCR on primary RB tumor samples (n = 30; including 17 invasive RB tumors and 13 noninvasive RB tumors). Detailed complementary alignment between 5' seed sequence of differentially expressed miRNAs and the sequence of target genes was determined. Based on minimum energy level and piCTAR scores, the gene targets were selected. Functional roles of these miRNA clusters were studied by using mimics in cultured RB (Y79, Weri Rb-1) cells in vitro. The gene targets (SYK and FASN) of the studied miRNAs were confirmed by qRT-PCR and western blot analysis. Cell proliferation and apoptotic studies were performed. RESULTS: Nearly 1948 miRNAs were identified in the in silico analysis, From this list, only 9 upregulated miRNAs (miR-146b-5p, miR-305, miR-663b, miR-299, miR-532-5p, miR-892b, miR-501, miR-142-5p, and miR-513b) and 10 downregulated miRNAs (miR-1254, miR-328, miR-133a, miR-1287, miR-1299, miR-375, miR-486-3p, miR-720, miR-98, and miR-122*) were found to be common with the RB serum miRNA profile. Downregulation of five miRNAs (miR-146b-5p, miR-532-5p, miR-142-5p, miR-328, and miR-486-3p) was confirmed experimentally. Predicted common oncogene targets (SYK and FASN) of miR-486-3p and miR-532-5p were evaluated for their mRNA and protein expression in these miRNA mimic-treated RB cells. Experimental overexpression of these miRNAs mediated apoptotic cell death without significantly altering the cell cycle in RB cells. CONCLUSION: Key miRNAs in RB pathogenesis were identified by an in silico approach. Downregulation of miR-486-3p and miR-532-5p in primary retinoblastoma tissues implicates their role in tumorigenesis. Prognostic and therapeutic potential of these miRNA was established by the miRNA mimic strategy.

Comparative Modeling and Molecular Dynamics Simulation of Substrate Binding in Human Fatty Acid Synthase: Enoyl Reductase and ß-Ketoacyl Reductase Catalytic Domains.

Fatty acid synthase (FASN, EC 2.3.1.85), is a multi-enzyme dimer complex that plays a critical role in lipogenesis. This lipogenic enzyme has gained importance beyond its physiological role due to its implications in several clinical conditions-cancers, obesity, and diabetes. This has made FASN an attractive pharmacological target. Here, we have attempted to predict the theoretical models for the human enoyl reductase (ER) and ß-ketoacyl reductase (KR) domains based on the porcine FASN crystal structure, which was the structurally closest template available at the time of this study. Comparative modeling methods were used for studying the structure-function relationships. Different validation studies revealed the predicted structures to be highly plausible. The respective substrates of ER and KR domains-namely, trans-butenoyl and ß-ketobutyryl-were computationally docked into active sites using Glide in order to understand the probable binding mode. The molecular dynamics simulations of the apo and holo states of ER and KR showed stable backbone root mean square deviation trajectories with minimal deviation. Ramachandran plot analysis showed 96.0% of residues in the most favorable region for ER and 90.3% for the KR domain, respectively. Thus, the predicted models yielded significant insights into the substrate binding modes of the ER and KR catalytic domains and will aid in identifying novel chemical inhibitors of human FASN that target these domains.

A molecular model of human Lysyl Oxidase (LOX) with optimal copper orientation in the catalytic cavity for induced fit docking studies with potential modulators.

Lysyl oxidase (LOX) is a copper dependent amine oxidase which catalyses the cross linking of collagen and elastin towards the maturation of extracellular matrix. The expression and activity of LOX is known to vary under pathological conditions such as tumorigenesis, hyperhomocysteinemia, copper deficiency diseases, pseudoexfoliation syndrome and proliferative diabetic retinopathy. Despite the implication of LOX in many diseases, there is inadequate information about its structure. Therefore, we describe a molecular model of Human Lysyl Oxidase (LOX) with optimal copper orientation in the catalytic cavity for induced fit docking studies with potential modulators. The predicted model was found to be highly plausible as per the stereochemistry checks. Further, Molecular Dynamics (MD) studies also inferred the stability of the predicted structure. We performed Induced Fit Docking (IFD) of LOX modulators to the predicted structure and also validated the molecular interactions in implicit solvent model by calculating Molecular Mechanics Generalized Born Surface Area (MMGBSA). The IFD results strongly reveal that aspartic acid residues in the catalytic cavity as the key players in establishing interactions with small molecules. The insights from this study will aid in better exploration of the structure-function relationship of LOX.

Molecular Insights on Post-chemotherapy Retinoblastoma by Microarray Gene Expression Analysis.

PURPOSE: Management of Retinoblastoma (RB), a pediatric ocular cancer is limited by drug-resistance and drug-dosage related side effects during chemotherapy. Molecular de-regulation in post-chemotherapy RB tumors was investigated. MATERIALS AND METHODS: cDNA microarray analysis of two post-chemotherapy and one pre-chemotherapy RB tumor tissues was performed, followed by Principle Component Analysis, Gene ontology, Pathway Enrichment analysis and Biological Analysis Network (BAN) modeling. The drug modulation role of two significantly up-regulated genes (p≤0.05) - Ect2 (Epithelial-cell-transforming-sequence-2), and PRAME (preferentially-expressed-Antigen-in-Melanoma) was assessed by qRT-PCR, immunohistochemistry and cell viability assays. RESULTS: Differential up-regulation of 1672 genes and down-regulation of 2538 genes was observed in RB tissues (relative to normal adult retina), while 1419 genes were commonly de-regulated between pre-chemotherapy and post- chemotherapy RB. Twenty one key gene ontology categories, pathways, biomarkers and phenotype groups harboring 250 differentially expressed genes were dys-regulated (EZH2, NCoR1, MYBL2, RB1, STAMN1, SYK, JAK1/2, STAT1/2, PLK2/4, BIRC5, LAMN1, Ect2, PRAME and ABCC4). Differential molecular expressions of PRAME and Ect2 in RB tumors with and without chemotherapy were analyzed. There was neither up- regulation of MRP1, nor any significant shift in chemotherapeutic IC50, in PRAME over-expressed versus non-transfected RB cells. CONCLUSION: Cell cycle regulatory genes were dys-regulated post-chemotherapy. Ect2 gene was expressed in response to chemotherapy-induced stress. PRAME does not contribute to drug resistance in RB, yet its nuclear localization and BAN information, points to its possible regulatory role in RB.

Biochemical changes accompanying apoptotic cell death in retinoblastoma cancer cells treated with lipogenic enzyme inhibitors.

Retinoblastoma (RB) is a malignant intra-ocular neoplasm that affects children (usually below the age of 5years). In addition to conventional chemotherapy, novel therapeutic strategies that target metabolic pathways such as glycolysis and lipid metabolism are emerging. Fatty acid synthase (FASN), a lipogenic multi-enzyme complex, is over-expressed in retinoblastoma cancer. The present study evaluated the biochemical basis of FASN inhibition induced apoptosis in cultured Y79 RB cells. FASN inhibitors (cerulenin, triclosan and orlistat) significantly inhibited FASN enzyme activity (P<0.05) in Y79 RB cells. This was accompanied by a decrease in palmitate synthesis (end-product depletion), and increased malonyl CoA levels (substrate accumulation). Differential lipid profile was biochemically estimated in neoplastic (Y79 RB) and non-neoplastic (3T3) cells subjected to FASN inhibition. The relative proportion of phosphatidyl choline to neutral lipids (triglyceride+total cholesterol) in Y79 RB cancer cells was found to be higher than the non-neoplastic cells, indicative of altered lipid distribution and utilization in tumor cells. FASN inhibitor treated Y79 RB and fibroblast cells showed decrease in the cellular lipids (triglyceride, cholesterol and phosphatidyl choline) levels. Apoptotic DNA damage induced by FASN inhibitors was accompanied by enhanced lipid peroxidation.

Fatty acid synthase inhibition induces differential expression of genes involved in apoptosis and cell proliferation in ocular cancer cells.

Fatty acid synthase (FASN), a lipogenic multienzyme complex, is overexpressed in the ocular cancer, retinoblastoma, and is strongly correlated with tumor invasion. Dietary nutrients are reported to exert anticancer effects through inhibition of lipid metabolism. Differential gene expression in cultured retinoblastoma cells induced by cerulenin, a chemical inhibitor of FASN, was evaluated by cDNA microarray analysis. Cerulenin treatment resulted in significant upregulation of cytochrome c (CYCS) by 1.2-fold, whereas S-phase kinase-associated protein-2 (SKP2), a negative regulator of cell cycle, and the lipid metabolic genes (PPARA, RXRA, and ACACB) were significantly downregulated by -1.59-, -1.8-, -1.83-, and -1.5-fold, respectively, in comparison with untreated cancer cells. The expressions of key differentially expressed genes were confirmed by quantitative real-time PCR. The altered expression of genes involved in cell proliferation, cell signaling, apoptosis, and cell cycle, correlated with the anticancer effects of cerulenin. FASN inhibition may thus be a potential strategy in retinoblastoma management.

The metabolic advantage of choline lactate in growth media: an experimental analysis with Staphylococcus lentus.

The metabolic effectiveness of choline lactate in the growth media was investigated relative to conventional carbon source for growing Staphylococcus lentus, a bacterial strain commonly used in bioremediation of industrial effluents and xenobiotic detoxification. Bacterial growth thermodynamics was determined by biocalorimetry. (13)C NMR and FTIR spectroscopic analyses traced the consumption of choline lactate at specific time intervals of bacterial growth. Under aerobic conditions, it is apparent that S. lentus initially metabolized lactate for its energy needs, while the choline cation of the ionic salt seemed to provide its C and N for biosynthetic intermediates for cell structure/function, in the growing bacterial colony. Urea accumulation after 40 h of bacterial growth was recorded. Possible metabolic trajectory of choline lactate consumed during S. lentus growth is suggested here. The theoretical estimation of heats of reaction for the proposed metabolic pathway (455 kJ/mol) was comparable with the experimentally obtained reaction enthalpy (435 kJ/mol), which further validated the proposed metabolic pathway. The biomass and energy profile of bacteria growth in choline media was found to be more favorable than in glucose media. The ionic liquid, choline lactate, offers a metabolically and energetically efficient carbon (and nitrogen) source for growing S. lentus.

Molecular deregulation induced by silencing of the high mobility group protein A2 gene in retinoblastoma cells.

AIM: To explore the molecular mechanisms deregulated by high mobility group protein A2 (HMGA2) gene silencing in retinoblastoma (RB) cells. METHODS: Synthetic anti-HMGA2 short interfering RNA (siRNA) was used to silence the HMGA2 gene in cultured Y79 RB cells that were subjected to whole genome microarray analysis. The expression of differentially regulated key genes was confirmed with quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) in post-silenced RB cell lines (Y79 and WERI Rb1). These deregulated genes were compared for their constitutive expression in primary RB tumors (n=10). Zymographic determination of matrix metalloproteinase (MMP) activity was performed in RB cells. A cell cycle assay and a proliferation assay were performed in post-transfected RB cells. RESULTS: HMGA2 gene silencing in cultured RB cells results in reduced cell proliferation and transition in the G1/S phase. The whole genome microarray analysis of HMGA2 silenced Y79 cells revealed overall upregulation of 1,132 genes (≥ 1.0 fold) and downregulation of 1,562 genes (≤ -1.0 fold). Specific quantitative pathway analysis of the deregulated genes (using Biointerpreter) revealed 150 upregulated genes and 77 downregulated genes (≥ 1.0 fold) involved in vital pathways, namely, mitogen-activated protein kinase, Janus kinase/signal transducers and activators of transcription, Ras pathway, Ras-induced extracellular signal-regulated protein kinases 1 and 2, and tumor protein p53. The differential expression of genes obtained from microarray analysis (Homo sapiens ELK1, member of ETS oncogene family [ELK1], Homo sapiens cyclin-dependent kinase 6 [CDK6], Homo sapiens E2F transcription factor 4, p107/p130-binding [E2F4], Homo sapiens G-2 and S-phase expressed 1 [GTSE1], Damage-regulated autophagy modulator [DRAM], Homo sapiens cadherin 1, type 1,E-cadherin (epithelial) [CDH1], Homo sapiens snail homolog 1 (Drosophila) [SNAI1], Homo sapiens matrix metallopeptidase 2 [MMP2], and Homo sapiens matrix metallopeptidase 9 [MMP9]) was confirmed with quantitative reverse-transcriptase polymerase chain reaction in post-silenced RB cells. Zymographic analysis revealed that the increase in MMP mRNA expression in the post-silenced RB cells did not correlate with corresponding enzyme activity. CONCLUSIONS: Our study revealed molecular regulatory changes induced by HMGA2 silencing in RB cancer cells, offering mechanistic insights into the anticancer potential. HMGA2 may be considered a promising candidate for gene silencing therapy in RB.

Microarray analysis and biochemical correlations of oxidative stress responsive genes in retinoblastoma.

PURPOSE: Oxidative stress, which refers to the biological damage caused by free radicals produced in excess of innate antioxidant defenses, is indicated in the ocular cancer retinoblastoma (RB). Here we have analysed the differential expression of oxidative stress responsive genes in oxidant-induced RB cells, and in RB tumor tissues. METHODS: The study included cultured RB cells, and four RB tumor tissues. The reactive oxygen species (ROS) levels in Y79 cells and the RB tumor induced by hydrogen peroxide were quantified by Dichlorofluorescein (DCF) fluorescence assay. We then analysed the gene expression profile of cultured RB cells induced with hydrogen peroxide (400 µM H(2)O(2) for 8 h) by microarray analysis, and the expression of select genes were validated in Y79 cells and RB tumor tissues by real-time PCR analysis. RESULTS: The oxidant-induced RB tumors showed an average increase in ROS levels of 44-fold compared to induced non-neoplastic donor retina. H(2)O(2)-induced RB cell line showed a 3-fold increase in ROS levels. Microarray analysis on RB cell line induced with H(2)O(2) showed differentially regulated genes involved in cellular processes such as: oxidative stress, angiogenesis, lipid metabolism, cell proliferation, and cell signaling pathways. Several up-regulated genes such as SOD, GPX, CAT, CDC25A, CREBBP, JUN, MMP-2, iNOS, CRYAA, RXRA, ACACB and HMGCR were validated by real-time PCR. These results corroborated with the gene expression analysis in RB tumor tissues. Relating the antioxidant gene expression with the clinico-pathologic features of the tumor tissues, we found that the tumor with invasion of choroid, optic nerve and retinal pigment epithelium, had relatively higher ROS levels and minimal antioxidant gene expression, when compared with the tumor with only choroidal invasion. CONCLUSIONS: The study suggests active involvement of redox signaling pathways in the pathogenesis of RB. Consideration of oxidative stress components in the clinical management of RB patients is emphasized.

Therapeutic and toxicologic evaluation of anti-lipogenic agents in cancer cells compared with non-neoplastic cells.

Fatty acid synthase (FASN), a multi-enzyme complex, is involved in lipid biosynthesis. FASN is over-expressed in different types of cancers and is being widely investigated for its role in cancer progression, diagnosis and therapy. Here, three inhibitors targeting different domains of FASN--cerulenin, triclosan and orlistat--were evaluated for their anti-proliferative efficacy in ocular cancer, retinoblastoma (RB) cells and their toxicity (if any) in normal cells. FASN inhibitors were tested in cultured retinoblastoma Y79 cells, normal fibroblast (3T3) and Müller glial (MIOM1) cells. Cell viability was determined by MTT-based assay, and IC(50) (50% inhibitory concentration) of the FASN inhibitors was calculated in neoplastic and non-neoplastic cells. The IC(50) after 48 and 96 hr of incubation with the three anti-FASN agents showed that cerulenin, triclosan and orlistat inhibited retinoblastoma cell proliferation in a dose- and time-dependent manner. The cancer cells exhibited differential dose- and time-dependent response/sensitivities to cerulenin, triclosan and orlistat. The 48-hr neoplastic IC(50) dosages were, however, not toxic to the normal cells. These findings were confirmed by phase-contrast microscopic assessment of cell morphology. Therapeutic index (TI) was calculated as a ratio of the IC(50) normal cells, to the IC(50) neoplastic cells. Relative to normal MIOM1 cells, TI was 9.18 for cerulenin, while 5.32 for triclosan and 1.72 for orlistat. The TI computed relative to 3T3 cells was 28.64, 7.10 and 2.58 for cerulenin, triclosan and orlistat, respectively. DNA fragmentation analysis suggests that FASN inhibitors induced apoptotic DNA damage in retinoblastoma cells. Thus, FASN inhibition can be an effective strategy in retinoblastoma therapy.

Comparative proteomic analysis of differentially expressed proteins in primary retinoblastoma tumors.

PURPOSE: To understand the disease mechanism and to identify the potential tumor markers that would help in therapeutics, comparative proteomic analysis of 29 retinoblastoma (RB) tumors was performed using 14 non-neoplastic retinas (age ranged from 45 to 89 years) as control tissues. EXPERIMENTAL DESIGN: 2-DE and MALDI-TOF-TOF MS/MS were used to identify differentially expressed proteins. RESULTS: Twenty-seven distinct differentially expressed proteins were identified, including 16 upregulated 11 downregulated proteins. Significantly, higher mRNA levels of apolipoprotein A1 (p<0.001), transferrin (TF; p<0.001), CRABP2 (p<0.001), α-crystallin A (CRYAA; p<0.001) were observed in RBs when compared with normal retinas and hence are consistent with the proteomic data. Immunohistochemistry was also performed for selected proteins on paraffin RB blocks to confirm protein expression. RB with invasion showed significantly higher expression by 2-DE-MS/MS analysis of CRABP2 (p<0.001), peroxiredoxin 6 (p=0.025), apolipoprotein A1 (p<0.001), recoverin (p<0.001). CONCLUSIONS AND CLINICAL RELEVANCE: Thus, this study provides a dynamic protein profile of RB tumors, which could provide clues to study the mechanisms of RB oncogenesis and possibly be developed as potential biomarkers for prognosis and therapy.

Protective effects of certoparin sodium, a low molecular weight heparin derivative, in experimental atherosclerosis.

BACKGROUND: The association of atherosclerosis and hypercholesterolemia is well known. Hypercholesterolemic diet-induced atherogenesis is a widely accepted experimental model that is amenable to exploration of both the disease as well as therapeutic interventions. We evaluated the role of low molecular weight heparin (LMWH) in modulating the early biochemical changes in atherogenesis. METHODS: Male Wistar rats (140 +/- 10 g) were fed an atherogenic diet comprising of normal rat chow supplemented with 4% cholesterol, 1% cholic acid and 0.5% thiouracil (CCT diet) for 2 weeks. While one of the CCT diet-fed group served as the untreated pathologic model, the other group received LMWH (Certoparin sodium, Troparin; 300 microg/day/rat s.c.) treatment, commencing on day 8 and continued for 1 week. RESULTS: Decreased concentrations of serum albumin and increased serum urea, uric acid and creatinine concentrations were normalized by LMWH treatment. The atherogenic diet induced abnormal rise in the activities of lactate dehydrogenase, aminotransferases and alkaline phosphatase, as well as the high serum cholesterol and triglyceride concentrations were restored to near control values in the treated group. LMWH administration prevented the hypertrophic cardiac histology and fatty changes in the liver in early atherogenesis. CONCLUSION: The present study encapsulates the early cellular abnormalities in the heart, liver and kidney tissues of atherogenic diet fed rats. Treatment with LMWH affords considerable protection to the tissues challenged by hypercholesterolemia, evidenced by its correction of lipemia and restoration of serum and tissue indices of injury, to normalcy. LMWH intervention minimized the atherogenic diet-induced histopathological lesions in heart, liver and kidney tissues.