Emerging roles for hyaluronidase in cancer metastasis and therapy

Hyaluronidases are a family of five human enzymes that have been differentially implicated in the progression of many solid tumor types, both clinically and in functional studies. Advances in the past five years have clarified many apparent contradictions, (1) by demonstrating that specific hyaluronidases have alternative substrates to hyaluronan (HA) or do not exhibit any enzymatic activity, (2) that high molecular weight HA polymers elicit signaling effects that are opposite those of the hyaluronidase-digested HA oligomers, and (3) that it is actually the combined overexpression of HA synthesizing enzymes with hyaluronidases that confers tumorigenic potential. This review examines the literature supporting these conclusions and discusses novel mechanisms by which hyaluronidases impact invasive tumor cell processes. In addition, a detailed structural and functional comparison of the hyaluronidases is presented with insights into substrate selectivity and potential for therapeutic targeting. Finally, technological advances in targeting hyaluronidase for tumor imaging and cancer therapy are summarized

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