Steroidal alkaloid solanidine impedes hypoxia-driven ATM phosphorylation to switch on anti-angiogenesis in lung adenocarcinoma.

PURPOSE: The declined oxygen tension in the cancer cell leads to the hypoxic adaptive response and favors establishment of tumor micro environment [TEM]. The complex TME consists of interwoven hypoxic HIF-1α and DNA damage repair ATM signaling. The ATM/HIF-1α phosphorylation switch on angiogenesis and abort apoptosis. Targeting this signaling nexus would be a novel therapeutic strategy for the treatment of cancer. BACKGROUND: Steroidal alkaloid solanidine is known for varied pharmacological role but with less molecular evidences. Our earlier findings on solanidine proven its anti-neoplastic activity by inducing apoptosis in lung cancer. In continued research, efforts have been made to establish the underlying molecular signaling in induction of DNA damage in prevailing hypoxic TME. METHODS: The solanidine induced DNA damage was assessed trough alkali COMET assay; signaling nexus and gene expression profile analysis through IB, qRT-PCR, Gelatin Zymography, IHC, IF and ELISA. Pathophysiological modulations assessed through tube formation, migration, invasion assays. Anti-angiogenic studies through CAM, rat aorta, matrigel assays and corneal neovascularization assay. Anti-tumor activity through in-vivo DLA ascites tumor model and LLC model. RESULTS: The results postulates, inhibition of hypoxia driven DDR proteins pATMser1981/pHIF-1αser696 by solanidine induces anti-angiogenesis. Systematic study of both non-tumorigenic and tumorigenic models in-vitro as well as in-vivo experimental system revealed the angio-regression mediated anticancer effect in lung cancer. These effects are due to the impeded expression of angiogenic mediators such as VEGF, MMP2&9 and inflammatory cytokines IL6 and TNFα to induce pathophysiological changes CONCLUSION: The study establishes new role of solanidine by targeting ATM/HIF-1α signaling to induce anti-angiogenesis for the first time. The study highlights the potentiality of plant based phytomedicine solanidine which can targets the multiple hallmarks of cancer by targeting interwoven signaling crosstalk. Such an approach through solanidine necessary to counteract heterogeneous complexity of cancer which could be nearly translated into drug.

Anti-neoplastic pharmacophore benzophenone-1 coumarin (BP-1C) targets JAK2 to induce apoptosis in lung cancer.

Reigning of the abnormal gene activation associated with survival signalling in lung cancer leads to the anomalous growth and therapeutic failure. Targeting specific cell survival signalling like JAK2/STAT3 nexus has become a major focus of investigation to establish a target specific treatment. The 2-bromobenzoyl-4-methylphenoxy-acetyl hydra acetyl Coumarin (BP-1C), is new anti-neoplastic agent with apoptosis inducing capacity. The current study was aimed to develop antitumor phramacophore, BP-1C as JAK2 specific inhibitor against lung neoplastic progression. The study validates and identifies the molecular targets of BP-1C induced cell death. Cell based screening against multiple cancer cell lines identified, lung adenocarcinoma as its specific target through promotion of apoptosis. The BP-1C is able to induce, specific hall marks of apoptosis and there by conferring anti-neoplastic activity. Validation of its molecular mechanism, identified, BP-1C specifically targets JAK2Tyr1007/1008 phosphorylation, and inhibits its downstream STAT3Tyr705 signalling pathway to induce cell death. As a consequence, modulation in Akt/Src survival signal and altered expression of interwoven apoptotic genes were evident. The results were reproducible in an in-vivo LLC tumor model and in-ovo xenograft studies. The computational approaches viz, drug finger printing confers, BP-1C as novel class JAK2 inhibitor and molecular simulations studies assures its efficiency in binding with JAK2. Overall, BP-1C is a novel JAK2 inhibitor with experimental evidence and could be effectively developed into a promising drug for lung cancer treatment.

In vivo Toxicity Studies on Gall Extracts of Terminalia chebula (Gaertn.) Retz. (Combretaceae).

UNLABELLED: The galls of Terminala chebula (Gaertn.) Retz. (Combretaceae) are used for the treatment of various diseases in folk medicine and has been found to posses anti-inflammatory, anti-bacterial, anti-helmintic, anti-tyrosinase, and anti-aging activities. Considering the ethano-botanical and diverse pharmacological applications of galls of T. chebula, in this study, we investigate the possible toxic effects of different gall extracts of T. chebula by Brine shrimp (Artemia salina) toxicity assay. The cytotoxicity test of leaf gall extracts (petroleum ether, chloroform, ethanol, and aqueous) of T. chebula was evaluated by Brine shrimp (A. salina) toxicity assay, which is based on the ability to kill laboratory cultured Artemia nauplii (animals eggs) and also total content of polyphenols, flavonoids with other qualitative phytochemical analysis of the extract were determined. It was observed that the petroleum ether extract was virtually nontoxic on the shrimps, and exhibited very low toxicity with LC50 value of 4356.76 µg/ml. Furthermore, the chloroform extract exhibited very low toxicity, giving LC50 value of 1462.2 µg/ml. On the other hand, the ethanol extract was very toxic to brine shrimps with LC50 value of 68.64 µg/ml. The ethanol extract had the highest total phenolic and flavonoid content of 136 ± 1.5 mg of gallic acid equivalent/g d.w and 113 ± 1.6 mg of quercetin equivalent/g d.w, respectively. The higher toxicity effect was positively correlated to the high content of total polyphenols/flavonoids in the extract. This significant lethality of different extracts to brine shrimp is an indicative of the presence of potent cytotoxic components which warrants further investigation. SUMMARY: The present study investigates the toxicity effect of different extracts of galls of T. chebulla, which would serve as an index for formulation of drugs for treatment of various diseases. Presumably, these activities could be attributed in part to the polyphenolic features of the extract, as there was a strong correlation of higher toxic effect with that of high total phenolic and flavonoids content in the ethanolic leaf gall extracts of T. chebula.

Evaluating the Anticancer Potential of Ethanolic Gall Extract of Terminalia chebula (Gaertn.) Retz. (Combretaceae).

UNLABELLED: Plants have been an important source for discovery of anticancer compounds. With the current decline in the number of new molecular entities from the pharmaceutical industry, novel anticancer agents are being sought from traditional medicines; therefore the anticancer efficacy of many plants that are used in traditional medicine is yet to be verified. The objective of the study was to evaluate the cytotoxic potential of ethanolic leaf gall extract of Terminalia chebula are evaluated against buffalo rat liver 3A, MCF-7 (Human mammary gland adenocarcinoma) and A-549 (Human lung cancer) cell lines. The cytotoxic effect of the ethanolic extract was evaluated by MTT assay. The extract was potent and effective in inducing cytotoxic effects in all the cell lines with an IC50 value of 305.18 ± 1.7 µg/mL, 643.13 ± 4.2 µg/mL, and 208.16 ± 3.7 µ/mL, respectively. The extract was more effective against A549 cell lines when compared to others. The presences of phenolics, triterpenoids, and flavonoids were identified in the extract. The extract showed total phenolic and flavonoid content of 478 ± 2.2 mg of gallic acid equivalent/g d.w and 538 ± 1.4 mg of quercetinequivalent/g d.w, respectively. This higher content of total phenolics and flavonoids found in the ethanolic extract was directly associated to higher cytotoxicity activity. CONCLUSION: The ethanolic leaf gall extract of T. chebula showed effective cytotoxic activities; which might be attributed to the phenolics/flavonoids present in higher concentration. Future work will be interesting to know the chemical composition of the extract and also better understand the mechanism of action of the constituents present in the extract to develop it as drug for therapeutic application. SUMMARY: The present investigation establishes the anticancer activities of T. chebula leaf gall extracts on BRL3A, MCF-7, and A-549 cells. Presumably, these activities could be attributed in part to the phenolics/flavanoids features of the extract that has been demonstrated to act as cytotoxic agents. The experimental evidence obtained in the laboratory model could provide a rationale for the traditional use of plant as a source of easily available effective anticancer agents to the people, particularly in developing countries.

Antioxidant activities of leaf galls extracts of Terminalia chebula (Gaertn.) Retz. (Combretaceae).

BACKGROUND: Free radicals are implicated in several metabolic diseases and the antioxidant therapy has gained an utmost importance in the treatment. The medicinal properties of plants have been investigated and explored for their potent antioxidant activities to counteract metabolic disorders. In this study, the chemical composition and free radical scavenging potential of leaf gall extracts (ethanol, petroleum ether, chloroform and aqueous) of Terminelia chebula is evaluated, which is extensively used in the preparation of traditiona medications to treat various metabolic diseases. METHODS: The presences of phenolics, flavonoids, triterpens, saponins, glycosides, phytosterols, reducing sugars were identified in the extracts according to standard procedures. The free radical scavenging activities of the extract were also analysed by standard procedures. RESULTS: The methanol extract had the highest total phenolic and flavonoid content. The antioxidant activities of leaf gall extracts were examined using diphenylpicrylhydrazyl (DPPH), Super oxide radical scavenging, Hydroxyl scavenging and ferric reducing power (FRAP) methods. In all the methods, the ethanolic extract showed higher free radical scavenging potential than all the other extracts. CONCLUSIONS: As the higher content of both total phenolics and flavonoids were found in the ethanolic extract, so the significantly high antioxidant activity can be positively correlated to the high content of total polyphenols/flavonoids of the ethanol extract. The results of this study confirm the folklore use of T. chebula leaves gall extracts as a natural antioxidant and justify its ethnobotanical use. Further, the results of antioxidant properties encourage the use of T. chebula leave gall extracts for medicinal health, functional food and nutraceutical applications.

Overexpression of ribosome binding protein 1 (RRBP1) in breast cancer.

The molecular events that lead to malignant transformation and subsequent metastasis of breast carcinoma include alterations in the cells at genome, transcriptome and proteome levels. In this study, we used publicly available gene expression databases to identify those candidate genes which are upregulated at the mRNA level in breast cancers but have not been systematically validated at the protein level. Based on an extensive literature search, we identified ribosome binding protein 1 (RRBP1) as a candidate that is upregulated at the mRNA level in five different studies but its protein expression had not been investigated. Immunohistochemical labeling of breast cancer tissue microarrays was carried out to determine the expression of RRBP1 in a large panel of breast cancers. We found that RRBP1 was overexpressed in 84% (177/219) of breast carcinoma cases tested. The subcellular localization of RRBP1 was mainly observed to be in the cytoplasm with intense staining in the perinuclear region. Our findings suggest that RRBP1 is an interesting molecule that can be further studied for its potential to serve as a breast cancer biomarker. This study also demonstrates how the integration of biological data from available resources in conjunction with systematic evaluation approaches can be successfully applied to clinical proteomics.

Overexpression of Kinesin Associated Protein 3 (KIFAP3) in Breast Cancer.

Gene expression profiling studies on breast cancer have generated catalogs of differentially expressed genes. However, many of these genes have not been investigated for their expression at the protein level. It is possible to systematically evaluate such genes in a high-throughput fashion for their overexpression at the protein level using breast cancer tissue microarrays. Our strategy involved integration of information from publicly available repositories of gene expression to prepare a list of genes upregulated at the mRNA level in breast cancer followed by curation of the published literature to identify those genes that were not tested for overexpression at the protein level. We identified Kinesin Associated Protein 3 (KIFAP3) as one such molecule for further validation at the protein level. Immunohistochemical labeling of KIFAP3 using breast cancer tissue microarrays revealed overexpression of KIFAP3 protein in 84% (240/285) of breast cancers indicating the utility of our integrated approach of combining computational analysis with experimental biology.

Gene Expression Profiling of Tuberculous Meningitis Co-infected with HIV.

Tuberculous meningitis (TBM) is a fatal form of Mycobacterium tuberculosis infection of the central nervous system (CNS). The similarities in the clinical and radiological findings in TBM cases with or without HIV make the diagnosis very challenging. Identification of genes, which are differentially expressed in brain tissues of HIV positive and HIV negative TBM patients, would enable better understanding of the molecular aspects of the infection and would also serve as an initial platform to evaluate potential biomarkers. Here, we report the identification of 796 differentially regulated genes in brain tissues of TBM patients co-infected with HIV using oligonucleotide DNA microarrays. We also performed immunohistochemical validation and confirmed the abundance of four gene products-glial fibrillary acidic protein (GFAP), serpin peptidase inhibitor, clade A member 3 (SERPINA3), thymidine phosphorylase (TYMP/ECGF1) and heat shock 70 kDa protein 8 (HSPA8). Our study paves the way for understanding the mechanism of TBM in HIV positive patients and for further validation of potential disease biomarkers.

Phytochemical analysis and hepatoprotective properties of Tinospora cordifolia against carbon tetrachloride-induced hepatic damage in rats.

The present study was conducted to evaluate the hepatoprotective activity of different extracts of Tinospora cordifolia against carbon tetrachloride (CCl4) induced liver damage in rats. The pet ether, ethanol and aqueous extracts of various parts of the plant such as leaf, stem and root were tested at the dose of 200mg/kg body weight orally using Wistar albino rats and Silymarin was given as reference standard. Ethanolic extract of all the parts showed significant hepatoprotective effect by reduction in serum enzymes alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and total bilirubin (TBL) in the selected model which is followed by aqueous and pet ether extracts. The chemical constituents reported from the plant belong to different classes such as alkaloids, flavanoids, glycosides, steroids, terpenoids, phenolics and saponins. The overall experimental results suggests that the biologically active phytoconstituents such as flavonoids, alkaloids present in the ethanolic extract of plant Tinospora cordifolia, may be responsible for the significant hepatoprotective activity. Therefore, results justify the use of Tinospora cordifolia as a hepatoprotective agent.

In vitro antioxidant activity of Holarrhena antidysenterica Wall. methanolic leaf extract.

Antioxidative potential of methanolic leaf extract of Holarrhena antidysenterica was evaluated using hydroxyl radical, superoxide anion scavenging and reducing power assays. The antioxidant activity of the methanol extract increased in a concentration-dependent manner. The extract showed significant reactive oxygen species (ROS) scavenging activity in all in vitro antioxidant assays and contained high level of total phenolic content.

NetPath: a public resource of curated signal transduction pathways.

We have developed NetPath as a resource of curated human signaling pathways. As an initial step, NetPath provides detailed maps of a number of immune signaling pathways, which include approximately 1,600 reactions annotated from the literature and more than 2,800 instances of transcriptionally regulated genes - all linked to over 5,500 published articles. We anticipate NetPath to become a consolidated resource for human signaling pathways that should enable systems biology approaches.

Prediction of candidate primary immunodeficiency disease genes using a support vector machine learning approach.

Screening and early identification of primary immunodeficiency disease (PID) genes is a major challenge for physicians. Many resources have catalogued molecular alterations in known PID genes along with their associated clinical and immunological phenotypes. However, these resources do not assist in identifying candidate PID genes. We have recently developed a platform designated Resource of Asian PDIs, which hosts information pertaining to molecular alterations, protein-protein interaction networks, mouse studies and microarray gene expression profiling of all known PID genes. Using this resource as a discovery tool, we describe the development of an algorithm for prediction of candidate PID genes. Using a support vector machine learning approach, we have predicted 1442 candidate PID genes using 69 binary features of 148 known PID genes and 3162 non-PID genes as a training data set. The power of this approach is illustrated by the fact that six of the predicted genes have recently been experimentally confirmed to be PID genes. The remaining genes in this predicted data set represent attractive candidates for testing in patients where the etiology cannot be ascribed to any of the known PID genes.

PathBuilder--open source software for annotating and developing pathway resources.

SUMMARY: We have developed PathBuilder, an open-source web application to annotate biological information pertaining to signaling pathways and to create web-based pathway resources. PathBuilder enables annotation of molecular events including protein-protein interactions, enzyme-substrate relationships and protein translocation events either manually or through automated importing of data from other databases. Salient features of PathBuilder include automatic validation of data formats, built-in modules for visualization of pathways, automated import of data from other pathway resources, export of data in several standard data exchange formats and an application programming interface for retrieving existing pathway datasets. AVAILABILITY: PathBuilder is freely available for download at http://pathbuilder.sourceforge.net/ under the terms of GNU lesser general public license (LGPL: http://www.gnu.org/copyleft/lesser.html). The software is platform independent and has been tested on Windows and Linux platforms. CONTACT: pandey@jhmi.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Human Protein Reference Database--2009 update.

Human Protein Reference Database (HPRD--http://www.hprd.org/), initially described in 2003, is a database of curated proteomic information pertaining to human proteins. We have recently added a number of new features in HPRD. These include PhosphoMotif Finder, which allows users to find the presence of over 320 experimentally verified phosphorylation motifs in proteins of interest. Another new feature is a protein distributed annotation system--Human Proteinpedia (http://www.humanproteinpedia.org/)--through which laboratories can submit their data, which is mapped onto protein entries in HPRD. Over 75 laboratories involved in proteomics research have already participated in this effort by submitting data for over 15,000 human proteins. The submitted data includes mass spectrometry and protein microarray-derived data, among other data types. Finally, HPRD is also linked to a compendium of human signaling pathways developed by our group, NetPath (http://www.netpath.org/), which currently contains annotations for several cancer and immune signaling pathways. Since the last update, more than 5500 new protein sequences have been added, making HPRD a comprehensive resource for studying the human proteome.

Human Proteinpedia: a unified discovery resource for proteomics research.

Sharing proteomic data with the biomedical community through a unified proteomic resource, especially in the context of individual proteins, is a challenging prospect. We have developed a community portal, designated as Human Proteinpedia (http://www.humanproteinpedia.org/), for sharing both unpublished and published human proteomic data through the use of a distributed annotation system designed specifically for this purpose. This system allows laboratories to contribute and maintain protein annotations, which are also mapped to the corresponding proteins through the Human Protein Reference Database (HPRD; http://www.hprd.org/). Thus, it is possible to visualize data pertaining to experimentally validated posttranslational modifications (PTMs), protein isoforms, protein-protein interactions (PPIs), tissue expression, expression in cell lines, subcellular localization and enzyme substrates in the context of individual proteins. With enthusiastic participation of the proteomics community, the past 15 months have witnessed data contributions from more than 75 labs around the world including 2710 distinct experiments, >1.9 million peptides, >4.8 million MS/MS spectra, 150,368 protein expression annotations, 17,410 PTMs, 34,624 PPIs and 2906 subcellular localization annotations. Human Proteinpedia should serve as an integrated platform to store, integrate and disseminate such proteomic data and is inching towards evolving into a unified human proteomics resource.

RAPID: Resource of Asian Primary Immunodeficiency Diseases.

Availability of a freely accessible, dynamic and integrated database for primary immunodeficiency diseases (PID) is important both for researchers as well as clinicians. To build a PID informational platform and also as a part of action to initiate a network of PID research in Asia, we have constructed a web-based compendium of molecular alterations in PID, named Resource of Asian Primary Immunodeficiency Diseases (RAPID), which is available as a worldwide web resource at http://rapid.rcai.riken.jp/. It hosts information on sequence variations and expression at the mRNA and protein levels of all genes reported to be involved in PID patients. The main objective of this database is to provide detailed information pertaining to genes and proteins involved in primary immunodeficiency diseases along with other relevant information about protein-protein interactions, mouse studies and microarray gene-expression profiles in various organs and cells of the immune system. RAPID also hosts a tool, mutation viewer, to predict deleterious and novel mutations and also to obtain mutation-based 3D structures for PID genes. Thus, information contained in this database should help physicians and other biomedical investigators to further investigate the role of these molecules in PID.

An evaluation of human protein-protein interaction data in the public domain.

BACKGROUND: Protein-protein interaction (PPI) databases have become a major resource for investigating biological networks and pathways in cells. A number of publicly available repositories for human PPIs are currently available. Each of these databases has their own unique features with a large variation in the type and depth of their annotations. RESULTS: We analyzed the major publicly available primary databases that contain literature curated PPI information for human proteins. This included BIND, DIP, HPRD, IntAct, MINT, MIPS, PDZBase and Reactome databases. The number of binary non-redundant human PPIs ranged from 101 in PDZBase and 346 in MIPS to 11,367 in MINT and 36,617 in HPRD. The number of genes annotated with at least one interactor was 9,427 in HPRD, 4,975 in MINT, 4,614 in IntAct, 3,887 in BIND and <1,000 in the remaining databases. The number of literature citations for the PPIs included in the databases was 43,634 in HPRD, 11,480 in MINT, 10,331 in IntAct, 8,020 in BIND and <2,100 in the remaining databases. CONCLUSION: Given the importance of PPIs, we suggest that submission of PPIs to repositories be made mandatory by scientific journals at the time of manuscript submission as this will minimize annotation errors, promote standardization and help keep the information up to date. We hope that our analysis will help guide biomedical scientists in selecting the most appropriate database for their needs especially in light of the dramatic differences in their content.

Effect of temperature and relative humidity on spinning behaviour of silkworm (Bombyx mori.L).

The rate of spinning of silkworm larva (Bombyx mori.L) was slow at 22 degrees C and fast at 38 degrees C. The time taken for completion of cocoon was longest at 98 +/- 2% and least at 40 +/- 2% RH. However, good quality cocoons were spun at 22 degrees C and 65 +/- 5% RH, hence it would be advantageous to maintain this temperature and relative humidity at the time of cocoon spinning in the case of CB race of silkworm.