In silico analysis of Human and Zebrafish ?-2 Adrenergic Receptors

?-2 adrenoceptors, belong to class of  Rhodopsin-like G-protein coupled receptors. Proteins of the G-protein coupled receptor (GPCR) family are involved in many pathophysiological conditions and hence are targets for various drug discovery methods. The current information on the structure of GPCRs is limited to few structures like Rhodopsin, ? adrenergic receptors, adenosine A2A receptors, Human Dopamine D3 and Chemokine receptor. In our study ?-2 adrenergic receptors of Human and Zebrafish were modeled using MODELLER with Human Dopamine D3 receptor (PDB ID: 3PBL) as template.  Through our modeling studies we have identified the critical role played by Proline residues (2.38, 2.59, 4.39, 4.59, 4.60, 7.50) of transmembrane helices and extracellular loop in stabilizing structural deviations in the transmembrane. Novel ligand binding residues S/T (6.56) and F (7.35) along with the positional significance of Y (3.28), Y (6.55) in regulating function were identified. Our models have shown that the Phenylalanine at 7.39 in TM7 can favourably interact with positively charged N-methyl group of the catecholamine ligands via hydrophobic contacts rather than 7.38 as reported previously. Furthermore, we are able to correctly show the orientation of Serine at 5.42 and 5.46 and discuss the relevance of residues at position 3.37 and 5.43 in the receptor regulation. We also demonstrate and propose that the orientation of V (2.61)/S should be taken into account in drug/ pharmacophore design specific for ?-2 adrenergic receptors. We believe that these findings will open new lead for ligand/ pharmacophore design, in silico leading further to experimental validation using Zebrafish as experimental model. Keywords: ?-2 adrenergic receptor, Zebrafish, ligand binding, residue conservation, homology modeling, ionic lock, toggle switch

Transcriptional Shift Identifies a Set of Genes Driving Breast Cancer Chemoresistance

Background Distant recurrences after antineoplastic treatment remain a serious problem for breast cancer clinical management, which threats patients’ life. Systemic therapy is administered to eradicate cancer cells from the organism, both at the site of the primary tumor and at any other potential location. Despite this intervention, a significant proportion of breast cancer patients relapse even many years after their primary tumor has been successfully treated according to current clinical standards, evidencing the existence of a chemoresistant cell subpopulation originating from the primary tumor.Methods/Findings To identify key molecules and signaling pathways which drive breast cancer chemoresistance we performed gene expression analysis before and after anthracycline and taxane-based chemotherapy and compared the results between different histopathological response groups (good-, mid- and bad-response), established according to the Miller & Payne grading system. Two cohorts of 33 and 73 breast cancer patients receiving neoadjuvant chemotherapy were recruited for whole-genome expression analysis and validation assay, respectively. Identified genes were subjected to a bioinformatic analysis in order to ascertain the molecular function of the proteins they encode and the signaling in which they participate. High throughput technologies identified 65 gene sequences which were over-expressed in all groups (P ≤ 0·05 Bonferroni test). Notably we found that, after chemotherapy, a significant proportion of these genes were over-expressed in the good responders group, making their tumors indistinguishable from those of the bad responders in their expression profile (P ≤ 0.05 Benjamini-Hochgerg`s method).Conclusions These data identify a set of key molecular pathways selectively up-regulated in post-chemotherapy cancer cells, which may become appropriate targets for the development of future directed therapies against breast cancer.Thanks are due to the Consejería de Economia, Innovación y Ciencia (CEIC) from the Junta de Andalucía and Fondo Europeo de Desarrollo Regional (FEDER)/Fondo de Cohesión Europeo (FSE) to financial support through the Programa Operativo FEDER/FSE de Andalucía 2007-2013 and the research project CTS-5350. The authors also acknowledge financial support by the PN de I+D+i 2006-2009/ISCIII/Ministerio de Sanidad, Servicios Sociales e Igualdad (Spain) and Fondo Europeo de Desarrollo Regional (FEDER) from the European Union, through the research project PI06/90388

Galiellalactone Inhibits Stem Cell-Like ALDH-Positive Prostate Cancer Cells

Galiellalactone is a potent and specific inhibitor of STAT3 signaling which has been shown to possess growth inhibitory effects on prostate cancer cells expressing active STAT3. In this study we aimed to investigate the effect of galiellalactone on prostate cancer stem cell-like cells. We explored the expression of aldehyde dehydrogenase (ALDH) as a marker for cancer stem cell-like cells in different human prostate cancer cell lines and the effects of galiellalactone on ALDH expressing (ALDH+) prostate cancer cells. ALDH+ subpopulations were detected and isolated from the human prostate cancer cell lines DU145 and long-term IL-6 stimulated LNCaP cells using ALDEFLUOR® assay and flow cytometry. In contrast to ALDH− cells, ALDH+ prostate cancer cells showed cancer stem cell-like characteristics such as increased self-renewing and colony forming capacity and tumorigenicity. In addition, ALDH+ cells showed an increased expression of putative prostate cancer stem cell markers (CD44 and integrin α2β1). Furthermore, ALDH+ cells expressed phosphorylated STAT3. Galiellalactone treatment decreased the proportion of ALDH+ prostate cancer cells and induced apoptosis of ALDH+ cells. The gene expression of ALDH1A1 was downregulated in vivo in galiellalactone treated DU145 xenografts. These findings emphasize that targeting the STAT3 pathway in prostate cancer cells, including prostate cancer stem cell-like cells, is a promising therapeutic approach and that galiellalactone is an interesting compound for the development of future prostate cancer drugs

Sustained proliferation in cancer: mechanisms and novel therapeutic targets

Proliferation is an important part of cancer development and progression. This is manifest by altered expression and/or activity of cell cycle related proteins. Constitutive activation of many signal transduction pathways also stimulates cell growth. Early steps in tumor development are associated with a fibrogenic response and the development of a hypoxic environment which favors the survival and proliferation of cancer stem cells. Part of the survival strategy of cancer stem cells may manifested by alterations in cell metabolism. Once tumors appear, growth and metastasis may be supported by overproduction of appropriate hormones (in hormonally dependent cancers), by promoting angiogenesis, by undergoing epithelial to mesenchymal transition, by triggering autophagy, and by taking cues from surrounding stromal cells. A number of natural compounds (e.g., curcumin, resveratrol, indole-3-carbinol, brassinin, sulforaphane, epigallocatechin-3-gallate, genistein, ellagitannins, lycopene and quercetin) have been found to inhibit one or more pathways that contribute to proliferation (e.g., hypoxia inducible factor 1, nuclear factor kappa B, phosphoinositide 3 kinase/Akt, insulin-like growth factor receptor 1, Wnt, cell cycle associated proteins, as well as androgen and estrogen receptor signaling). These data, in combination with bioinformatics analyses, will be very important for identifying signaling pathways and molecular targets that may provide early diagnostic markers and/or critical targets for the development of new drugs or drug combinations that block tumor formation and progression

Molecular marks for epigenetic identification of developmental and cancer stem cells

Epigenetic regulations of genes by reversible methylation of DNA (at the carbon-5 of cytosine) and numerous reversible modifications of histones play important roles in normal physiology and development, and epigenetic deregulations are associated with developmental disorders and various disease states, including cancer. Stem cells have the capacity to self-renew indefinitely. Similar to stem cells, some malignant cells have the capacity to divide indefinitely and are referred to as cancer stem cells. In recent times, direct correlation between epigenetic modifications and reprogramming of stem cell and cancer stem cell is emerging. Major discoveries were made with investigations on reprogramming gene products, also known as master regulators of totipotency and inducer of pluoripotency, namely, OCT4, NANOG, cMYC, SOX2, Klf4, and LIN28. The challenge to induce pluripotency is the insertion of four reprogramming genes (Oct4, Sox2, Klf4, and c-Myc) into the genome. There are always risks of silencing of these genes by epigenetic modifications in the host cells, particularly, when introduced through retroviral techniques. In this contribution, we will discuss some of the major discoveries on epigenetic modifications within the chromatin of various genes associated with cancer progression and cancer stem cells in comparison to normal development of stem cell. These modifications may be considered as molecular signatures for predicting disorders of development and for identifying disease states

Wnt pathway activity in breast cancer sub-types and stem-like cells

INTRODUCTION: Wnt signalling has been implicated in stem cell regulation however its role in breast cancer stem cell regulation remains unclear. METHODS: We used a panel of normal and breast cancer cell lines to assess Wnt pathway gene and protein expression, and for the investigation of Wnt signalling within stem cell-enriched populations, mRNA and protein expression was analysed after the selection of anoikis-resistant cells. Finally, cell lines and patient-derived samples were used to investigate Wnt pathway effects on stem cell activity in vitro. RESULTS: Wnt pathway signalling increased in cancer compared to normal breast and in both cell lines and patient samples, expression of Wnt pathway genes correlated with estrogen receptor (ER) expression. Furthermore, specific Wnt pathway genes were predictive for recurrence within subtypes of breast cancer. Canonical Wnt pathway genes were increased in breast cancer stem cell-enriched populations in comparison to normal breast stem cell-enriched populations. Furthermore in cell lines, the ligand Wnt3a increased whilst the inhibitor DKK1 reduced mammosphere formation with the greatest inhibitory effects observed in ER+ve breast cancer cell lines. In patient-derived metastatic breast cancer samples, only ER-ve mammospheres were responsive to the ligand Wnt3a. However, the inhibitor DKK1 efficiently inhibited both ER+ve and ER-ve breast cancer but not normal mammosphere formation, suggesting that the Wnt pathway is aberrantly activated in breast cancer mammospheres. CONCLUSIONS: Collectively, these data highlight differential Wnt signalling in breast cancer subtypes and activity in patient-derived metastatic cancer stem-like cells indicating a potential for Wnt-targeted treatment in breast cancers