Overexpression of CD44 accompanies acquired tamoxifen resistance in MCF7 cells and augments their sensitivity to the stromal factors, heregulin and hyaluronan

Background: Acquired resistance to endocrine therapy in breast cancer is a significant problem with relapse being associated with local and/or regional recurrence and frequent distant metastases. Breast cancer cell models reveal that endocrine resistance is accompanied by a gain in aggressive behaviour driven in part through altered growth factor receptor signalling, particularly involving erbB family receptors. Recently we identified that CD44, a transmembrane cell adhesion receptor known to interact with growth factor receptors, is upregulated in tamoxifen-resistant (TamR) MCF7 breast cancer cells. The purpose of this study was to explore the consequences of CD44 upregulation in an MCF7 cell model of acquired tamoxifen resistance, specifically with respect to the hypothesis that CD44 may influence erbB activity to promote an adverse phenotype. Methods: CD44 expression in MCF7 and TamR cells was assessed by RT-PCR, Western blotting and immunocytochemistry. Immunofluorescence and immunoprecipitation studies revealed CD44-erbB associations. TamR cells (± siRNA-mediated CD44 suppression) or MCF7 cells (± transfection with the CD44 gene) were treated with the CD44 ligand, hyaluronon (HA), or heregulin and their in vitro growth (MTT), migration (Boyden chamber and wound healing) and invasion (Matrigel transwell migration) determined. erbB signalling was assessed using Western blotting. The effect of HA on erbB family dimerisation in TamR cells was determined by immunoprecipitation in the presence or absence of CD44 siRNA. Results: TamR cells overexpressed CD44 where it was seen to associate with erbB2 at the cell surface. siRNA-mediated suppression of CD44 in TamR cells significantly attenuated their response to heregulin, inhibiting heregulin-induced cell migration and invasion. Furthermore, TamR cells exhibited enhanced sensitivity to HA, with HA treatment resulting in modulation of erbB dimerisation, ligand-independent activation of erbB2 and EGFR and induction of cell migration. Overexpression of CD44 in MCF7 cells, which lack endogenous CD44, generated an HA-sensitive phenotype, with HA-stimulation promoting erbB/EGFR activation and migration. Conclusions: These data suggest an important role for CD44 in the context of tamoxifen-resistance where it may augment cellular response to erbB ligands and HA, factors that are reported to be present within the tumour microenvironment in vivo. Thus CD44 may present an important determinant of breast cancer progression in the setting of endocrine resistance

Structure of casein micelles in milk protein concentrate powders via small angle X-ray scattering

The stability of milk protein concentrate (MPC) powders and their solubility in water is presumed to depend on the interfacial and internal structure of the casein micelle. This study reports the internal micellar structure for MPC powder for the first time. We have investigated the nanostructure of MPC powders and of dilute solutions thereof using small-angle X-ray scattering (SAXS). In addition, we have measured the scattering from the primary components of MPC in their powder and solution state to enable an assessment of their contribution to the overall scattering from the whole system. The interfacial and internal structural detail of the casein micelle is still an area of debate and we have considered the two popular models for casein micelles, namely the submicelle and nanocluster models; we find that the latter adequately describes the observed experimental results. The scattering curve for the powders may be described by three characteristic regions. The first region up to 0.03 A-1 is interpreted to correspond to the scattering from a sharp and smooth interface. The second inflexion point, observed at [similar]0.045 A-1, may be attributed to a mean intercluster correlation length of colloidal calcium phosphate (CCP) nanoclusters distributed within casein micelles. This is the first time such a feature has been observed in the powder state but its presence is reminiscent of a similar feature observed previously with solvent contrast variation small-angle neutron scattering. We interpret that its presence here is due to an enhancement of the scattering contrast by virtue of the removal of water from the system (and replaced by air) combined with an increase in particle density due to drying. The third inflexion at 0.1 A-1 may be interpreted, in common with other authors, as a signature of colloidal calcium phosphate nanoparticles.© 2011, Royal Society of Chemistr

Heat-induced changes in the properties of modified skim milks with different casein to whey protein ratios

Published online by Cambridge University Press: 12 December 2014The heat-induced changes in pH, Ca activity and viscosity after heating at 90 °C for 10 min of five modified skim milks were studied as a function of the initial pH of the milks at 25 °C. The milks had (i) different ratios of casein : whey protein (0.03, 1.74, 3.97, 5.27 and 7.25), (ii) the same total solids concentration (9% w/w) and (iii) prior to the adjustment of the pH, similar values of pH (6.67-6.74), concentration of serum calcium, and calcium activity, suggesting that the sera have similar mineral composition. The total protein concentrations of the milks differ (2.8-4.0%, w/w). The pH decrease in situ upon heating from 25-90 °C was similar for all the modified skim milks with the same starting pH, suggesting that the pH changes to milk on heating were primarily mediated by the initial mineral composition of the serum and were unaffected by the casein : whey protein ratio or the total protein content of the milk. The heat-induced changes in pH and calcium activity were largely reversible on cooling. The two milks with the lowest ratios of casein to whey protein gelled on heating to 90 °C for 10 min and cooling to 25 °C when the pH was adjusted to pH = 6.2 prior to heating. The viscosities of all other milks with casein to whey protein ratio of 3.97, 5.27 and 7.25 and/or pH ≥6.7 prior to heating did not change significantly. The effect of casein : whey protein ratio and the pH are the dominant factors in controlling the susceptibility to thickening of the milks on heating in this study.Mandeep Jeswan Singh, Jayani Chandrapala,Punsandani Udabage, Ian McKinnon and Mary Ann Augusti

Reconstitution properties of micellar casein powder: Effects of composition and storage

A problem associated with micellar casein (MC) powders is their poor reconstitution properties. In this study, we prepared MCs with different salt and protein compositions and having different reconstitutabilities directly after production. Reconstitutability further decreased during storage at 30 degrees C. Differential scanning calorimetry, infrared spectroscopy, and small angle X-ray scattering showed that the powders were very similar on a molecular or sub-micellar level, indicating that the loss of reconstitutability is probably controlled by higher order structural changes, such as cross-linking between casein micelles, possibly involving intermolecular beta-sheet formation. The reconstitutability of MC could be improved by adding sodium caseinate to the concentrated milk before spray drying. This novel approach to improve reconstitutability can easily be incorporated into the existing processing protocol. We propose two possible mechanisms for the protecting effect of the non-micellar caseins.(C) 2011 Elsevier Ltd. All rights reserved

CD44 enhances invasion of basal-like breast cancer cells by upregulating serine protease and collagen-degrading enzymatic expression and activity

INTRODUCTION: Basal-like breast cancers (BL-BCa) have the worst prognosis of all subgroups of this disease. Hyaluronan (HA) and the HA receptor CD44 have a long-standing association with cell invasion and metastasis of breast cancer. The purpose of this study was to establish the relation of CD44 to BL-BCa and to characterize how HA/CD44 signaling promotes a protease-dependent invasion of breast cancer (BrCa) cells. METHODS: CD44 expression was determined with immunohistochemistry (IHC) analysis of a breast cancer tissue microarray (TMA). In vitro experiments were performed on a panel of invasive BL-BCa cell lines, by using quantitative polymerase chain reaction (PCR), immunoblotting, protease activity assays, and invasion assays to characterize the basis of HA-induced, CD44-mediated invasion. RESULTS: Expression of the hyaluronan (HA) receptor CD44 associated with the basal-like subgroup in a cohort of 141 breast tumor specimens (P = 0.018). Highly invasive cells of the representative BL-BCa cell line, MDA-MB-231 (MDA-MB-231Hi) exhibited increased invasion through a basement membrane matrix (Matrigel) and collagen. In further experiments, HA-induced promotion of CD44 signaling potentiated expression of urokinase plasminogen activator (uPA) and its receptor uPAR, and underpinned an increased cell-associated activity of this serine protease in MDA-MB-231Hi and a further BL-BCa cell line, Hs578T cells. Knockdown of CD44 attenuated both basal and HA-stimulated uPA and uPAR gene expression and uPA activity. Inhibition of uPA activity by using (a) a gene-targeted RNAi or (b) a small-molecule inhibitor of uPA attenuated HA-induced invasion of MDA-MB-231Hi cells through Matrigel. HA/CD44 signaling also was shown to increase invasion of MDA-MB-231 cells through collagen and to potentiate the collagen-degrading activity of MDA-MB-231Hi cells. CD44 signaling was subsequently shown to upregulate expression of two potent collagen-degrading enzymes, the cysteine protease cathepsin K and the matrix metalloprotease MT1-MMP. RNAi- or shRNA-mediated depletion of CD44 in MDA-MB-231Hi cells decreased basal and HA-induced cathepsin K and MT1-MMP expression, reduced the collagen-degrading activity of the cell, and attenuated cell invasion through collagen. Pharmacologic inhibition of cathepsin K or RNAi-mediated depletion of MT1-MMP also attenuated MDA-MB-231Hi cell invasion through collagen. CONCLUSION: HA-induced CD44 signaling increases a diverse spectrum of protease activity to facilitate the invasion associated with BL-BCa cells, providing new insights into the molecular basis of CD44-promoted invasion

Protein nanotechnology

Cite this entry as: Yaradoddi J.S., Kontro M.H., Ganachari S.V., Sulochana M.B., Agsar D. (2019) Protein Nanotechnology. In: Martínez L., Kharissova O., Kharisov B. (eds) Handbook of Ecomaterials. Springer, Cham Publisher Name: Springer, Cham DOI: https://doi.org/10.1007/978-3-319-68255-6_192 Print ISBN: 978-3-319-68254-9 Online ISBN: 978-3-319-68255-6 First Online: 14 February 2019Medical management should be well-preserved; in particular, a fast, easy, and cheap diagnosis. Sometimes, a RNA and DNA nanobio-based diagnostic may not provide precise data with regard to specific disorders. Therefore, some quantifiable protein information and molecular folding are required for the analysis of such disorders. Proteins at minute concentrations are typically undetectable under normal circumstances nowadays, and can be measured and quantified using protein nanotechnology methods. On the other hand, protein machinery carry out tasks that are unsafe for cell behavior, comprising DNA duplication, intracellular carriage, ion pumps, and cellular motility. They have changed with unbelievable multiplicity, precision, efficacy, and a substantial number of studies in contemporary biology have been intended to expose the vital mechanisms or processes of their primary function. This chapter also emphasizes the recent developments in protein nanotechnology, with a special focus on molecular cytoskeletal motors, dyneins, myosins, and kinesins. They constitute a subcategory of protein machineries; they have distinguished properties and are able to convert biochemical energy to work automatically.Peer reviewe