The in vitro influences of epidermal growth factor and heregulin-β1 on the efficacy of trastuzumab used in Her-2 positive breast adenocarcinoma

BACKGROUND: Human epidermal growth factor receptor-2 (Her-2) is over expressed in approximately 25-30% of all primary breast tumors resulting in a distinctive breast cancer subtype associated with a poor prognosis and a decrease in overall survival. Trastuzumab (Herceptin®), an anti-Her-2 monoclonal antibody, has dramatically altered the prognosis of Her-2 positive breast cancer. Trastuzumab is, however, associated with primary and acquired resistance. AIM AND METHODS: To investigate the in-vitro effects of trastuzumab on cell viability (tetrazolium conversion assay), cell cycling (propidium iodide staining), apoptosis (executioner caspases and annexin-V) and relative surface Her-2 receptor expression (anti-Her-2 affibody molecule) in Her-2-positive (SK-Br-3) and oestrogen receptor positive (MCF-7) breast adenocarcinoma cells and to determine potential augmentation of these effects by two endogenous ligands, epidermal growth factor (EGF) and heregulin-β1 (HRG- β1). RESULTS: Cell viability was decreased in SK-Br-3 cells by exposure to trastuzumab. This was associated with G1 accumulation and decreased relative surface Her-2 receptor density, supporting the cytostatic nature of trastuzumab in vitro. SK-Br-3 cells exposed to EGF and heregulin-β1 produced differential cell responses alone and in combination with trastuzumab, in some instances augmenting cell viability and cell cycling. Relative surface Her-2 receptor density was reduced substantially by trastuzumab, EGF and heregulin-β1. These reductions were amplified when ligands were used in combination with trastuzumab. CONCLUSION: Cell type specific interactions of endogenous ligands appear to be dependent on absolute Her-receptor expression and cross activation of signaling pathways. This supports the notion that receptor density of Her-family members and multiplicity of growth ligands are of mutual importance in breast cancer cell proliferation and therefore also in resistance associated with trastuzumab.The authors would like to acknowledge Roche Pharmaceuticals for the donation of trastuzumab and the Cancer Association of South Africa (CANSA) and the Research and Development Programme (RDP), University of Pretoria, for providing funding.http://www.cancerci.com/content/13/1/97am2014ay201

Human epidermal growth factor receptor 2-positive breast cancer : which cytotoxic agent best complements trastuzumab's efficacy in vitro?

INTRODUCTION: Despite trastuzumab having enhanced selectivity for human epidermal growth factor receptor 2 (HER-2) overexpressing breast cancer cells, treatment is hampered by interindividual variation and tumors with high mitogenic potential. The lack of significant clinical benefit in certain patient cohorts suggests that HER-2 expression is ineffective as a sole prognostic indicator of response to therapy. Therefore, optimizing the clinical role of trastuzumab in drug combinations remains critical for clinical success. AIM: To investigate the effects of trastuzumab in combination with either doxorubicin or geldanamycin on in vitro cell viability, cell cycling, apoptosis and relative HER-2 expression in HER-2-positive (SK-BR-3) and estrogen receptor-positive (MCF-7) breast adenocarcinoma models. RESULTS: HER-2-rich SK-BR-3 cells demonstrated a greater sensitivity to the effects of doxorubicin than MCF-7 cells. Concurrent trastuzumab exposure resulted in a further reduction in cell viability. This decreased cell viability induced by doxorubicin was associated with activation of executioner caspases as well as with alterations in cell-cycle kinetics, primarily promoting S-phase accumulation. Doxorubicin had no effect on surface HER-2 density expression. Geldanamycin reduced cell viability significantly greater in SK-BR-3 than MCF-7 cells, and was associated with G2 cell-cycle accumulation. The addition of trastuzumab did not augment these effects. Geldanamycin promoted substantial reductions in relative surface HER-2 density in SK-BR-3 cells. CONCLUSION: The in vitro data supported the rationale for using doxorubicin in trastuzumab-based therapies. Therefore, despite the incidence of cardiotoxicity, doxorubicin could retain a fundamental role in treating HER-2-positive breast cancer. While geldanamycin is a potent cytotoxic agent, its concurrent use with trastuzumab requires further research into the transient or permanent nature of alterations in HER-2 status in cell progeny.The authors would like to acknowledge Roche Pharmaceuticals for the kind donation of trastuzumab and the Cancer Association of South Africa (CANSA) as well as the Research and Development Program (RDP), University of Pretoria, for providing their generous funding, and to Dr AD Cromarty and Dr JJ van Tonder without which this project would not have been possible.http://www.dovepress.comam201

A proteomic time course through the differentiation of human induced pluripotent stem cells into hepatocyte-like cells.

Numerous in vitro models endeavour to mimic the characteristics of primary human hepatocytes for applications in regenerative medicine and pharmaceutical science. Mature hepatocyte-like cells (HLCs) derived from human induced pluripotent stem cells (hiPSCs) are one such in vitro model. Due to insufficiencies in transcriptome to proteome correlation, characterising the proteome of HLCs is essential to provide a suitable framework for their continual optimization. Here we interrogated the proteome during stepwise differentiation of hiPSCs into HLCs over 40 days. Whole cell protein lysates were collected and analysed using stabled isotope labelled mass spectrometry based proteomics. Quantitative proteomics identified over 6,000 proteins in duplicate multiplexed labelling experiments across two different time course series. Inductive cues in differentiation promoted sequential acquisition of hepatocyte specific markers. Analysis of proteins classically assigned as hepatic markers demonstrated trends towards maximum relative abundance between differentiation day 30 and 32. Characterisation of abundant proteins in whole cells provided evidence of the time dependent transition towards proteins corresponding with the functional repertoire of the liver. This data highlights how far the proteome of undifferentiated precursors have progressed to acquire a hepatic phenotype and constructs a platform for optimisation and improved maturation of HLC differentiation

Assessing adherence to the 2010 antiretroviral guidelines in the antiretroviral roll-out clinic at 1 Military Hospital, South Africa : a retrospective, cross-sectional study

BACKGROUND. HIV research is a therapeutic area for which well-defined population-specific treatment and prophylaxis guidelines exist. However, there are limited objective, evidence-based data for assessing adherence to these guidelines. OBJECTIVE. To conduct a retrospective, cross-sectional study of adult HIV-infected patients receiving treatment at the antiretroviral (ARV) roll-out clinic of the Infectious Diseases Clinic Pharmacy at 1 Military Hospital (1MH) over a period of 3 years to assess clinicians’ adherence to the 2010 ARV guidelines. METHODS. Pharmacy files from the pool of adult patients receiving treatment at the ARV roll-out clinic between 1 April 2009 and 31 March 2012 were selected. Variables used to establish adherence were assessed through evaluation of pharmacy scripts and laboratory tests. RESULTS. In accordance with the ARV guidelines, we found a switch in the first-line regimen from stavudine to tenofovir during the period following implementation. There was no difference in baseline blood tests conducted, suggesting that clinicians were recommending a standardised test panel. Notably, similar blood tests were routinely done during follow-up visits, despite no indication for doing so. While the number of blood tests was found to decrease over time, the type of blood tests requested for specific treatment regimens was not in accordance with the ARV guidelines. CONCLUSION. We used an evidence-based approach to critically assess variations from the delineated ARV guidelines. Adherence to clinical guidelines at 1MH, while demonstrating improvement in patient outcomes, highlighted the need for increased vigilance in monitoring failure of prescribers to comply with ARV guidelines.http://www.samj.org.zaam201

Continual proteomic divergence of HepG2 cells as a consequence of long-term spheroid culture

Three-dimensional models are considered a powerful tool for improving the concordance between in vitro and in vivo phenotypes. However, the duration of spheroid culture may infuence the degree of correlation between these counterparts. When using immortalised cell lines as model systems, the assumption for consistency and reproducibility is often made without adequate characterization or validation. It is therefore essential to defne the biology of each spheroid model by investigating proteomic dynamics, which may be altered relative to culture duration. As an example, we assessed the infuence of culture duration on the relative proteome abundance of HepG2 cells cultured as spheroids, which are routinely used to model aspects of the liver. Quantitative proteomic profling of whole cell lysates labelled with tandem-mass tags was conducted using liquid chromatographytandem mass spectrometry (LC–MS/MS). In excess of 4800 proteins were confdently identifed, which were shared across three consecutive time points over 28 days. The HepG2 spheroid proteome was divergent from the monolayer proteome after 14 days in culture and continued to change over the successive culture time points. Proteins representing the recognised core hepatic proteome, cell junction, extracellular matrix, and cell adhesion proteins were found to be continually modulated.The National Research Foundation (NRF) Tuthuka PhD Track Funding Scheme, the NRF Innovation Masters and Doctoral Scholarship Program, University of Pretoria Doctoral Bursary as well as the NRF National Equipment Program.http://www.nature.com/srep/index.htmlpm2022PharmacologyPhysiolog

A draft map of the mouse pluripotent stem cell spatial proteome.

Knowledge of the subcellular distribution of proteins is vital for understanding cellular mechanisms. Capturing the subcellular proteome in a single experiment has proven challenging, with studies focusing on specific compartments or assigning proteins to subcellular niches with low resolution and/or accuracy. Here we introduce hyperLOPIT, a method that couples extensive fractionation, quantitative high-resolution accurate mass spectrometry with multivariate data analysis. We apply hyperLOPIT to a pluripotent stem cell population whose subcellular proteome has not been extensively studied. We provide localization data on over 5,000 proteins with unprecedented spatial resolution to reveal the organization of organelles, sub-organellar compartments, protein complexes, functional networks and steady-state dynamics of proteins and unexpected subcellular locations. The method paves the way for characterizing the impact of post-transcriptional and post-translational modification on protein location and studies involving proteome-level locational changes on cellular perturbation. An interactive open-source resource is presented that enables exploration of these data.The authors thank Andreas Hühmer, Philip Remes, Jesse Canterbury and Graeme McAlister of Thermo Fisher Scientific, San Jose, CA, USA, for their advice regarding operation of the Orbitrap Fusion. We also thank Mike Deery for assistance with checking sample integrity on the mass spectrometers in the Cambridge Centre for Proteomics on equipment purchased via a Wellcome Trust grant (099135/Z/12/Z ), and Brian Hendrich of the Wellcome Trust-MRC Stem Cell Institute in Cambridge and Sean Munro of the MRC Laboratory of Molecular Biology in Cambridge for insightful comments about the data. AC was supported by BBSRC grant (BB/D526088/1). C.M.M. and L.G. were supported by European Union 7th Framework Program (PRIMEXS project, grant agreement number 262067), L.M.B was supported by a BBSRC Tools and Resources Development Fund (Award BB/K00137X/1), and P.C.H. was supported by an ERC Advanced Investigator grant to A.M.A. A.G. was funded through the Alexander S. Onassis Public Benefit Foundation, the Foundation for Education and European Culture (IPEP) and the Embiricos Trust Scholarship of Jesus College Cambridge. T.H. was supported by Commonwealth Split Site PhD Scholarship. T.N. was supported by an ERASMUS Placement scholarshipThis is the final version of the article. It was first available from NPG via http://dx.doi.org/10.1038/ncomms999

Spatiotemporal proteomic profiling of the pro-inflammatory response to lipopolysaccharide in the THP-1 human leukaemia cell line.

Protein localisation and translocation between intracellular compartments underlie almost all physiological processes. The hyperLOPIT proteomics platform combines mass spectrometry with state-of-the-art machine learning to map the subcellular location of thousands of proteins simultaneously. We combine global proteome analysis with hyperLOPIT in a fully Bayesian framework to elucidate spatiotemporal proteomic changes during a lipopolysaccharide (LPS)-induced inflammatory response. We report a highly dynamic proteome in terms of both protein abundance and subcellular localisation, with alterations in the interferon response, endo-lysosomal system, plasma membrane reorganisation and cell migration. Proteins not previously associated with an LPS response were found to relocalise upon stimulation, the functional consequences of which are still unclear. By quantifying proteome-wide uncertainty through Bayesian modelling, a necessary role for protein relocalisation and the importance of taking a holistic overview of the LPS-driven immune response has been revealed. The data are showcased as an interactive application freely available for the scientific community

The effects of selected therapeutic agents on cell cytotoxicity and Her-2 receptor expression using culturedbreast adenocarcinoma models

Introduction: Epidemiological studies suggest that at least 1 in 29 South African women will be diagnosed with breast cancer in their lifetime. Breast cancer is not a single disease. The heterogeneity of breast cancer results in four distinct molecular subtypes including aggressive human epidermal growth factor receptor-2 (Her-2) positive, where Her-2 receptors are overexpressed. Trastuzumab (Herceptin®), is a recombinant, humanized, anti-Her-2 monoclonal antibody that specifically targets subdomain IV of the extracellular domain of the Her-2 receptor and has dramatically altered the prognosis of Her-2 positive breast cancer. Trastuzumab is, however, associated with problems such as primary and acquired resistance, which has prompted investigation into improving its efficacy. Aim: To investigate the ability of selected therapeutic agents to alter in-vitro cell viability, cell cycling, apoptosis and Her-2 expression in models of Her-2-positive and oestrogen receptor positive, Her-2 negative breast adenocarcinoma and bring about an alteration in the efficacy of trastuzumab. Methods: MCF-7 cells which retain the ability to process oestrogen, and SK-Br-3 cells which overexpress Her-2 gene products were used. Cells were exposed to trastuzumab, aspirin, calcipotriol, doxorubicin, epidermal growth factor (EGF-human), geldanamycin, heregulin-β1 and β-oestradiol as single agents and in combination with trastuzumab. Research methodologies included tetrazolium conversion assay for cell viability, AMC-substrate cleavage and annexin-V for apoptosis, propidium iodide staining for cell cycle analysis and anti-Her-2 affibody molecule for relative Her-2 receptor density. Results: Cell survival of 95.39% (±2.69) for MCF-7 cells and 74.17% (±1.60) for SK-Br-3 cells was observed following trastuzumab (100 μg/ml) exposure. Trastuzumab resulted in statistically significant G1 phase accumulation in MCF-7 cells at 72 hours and in SK-Br-3 cells from 24 hours. Furthermore, trastuzumab decreased relative Her-2 receptor density in SK-Br-3 cells by approximately 35% by 24 hours but had no effect in MCF-7 cells. The anti-proliferative effects of trastuzumab were abrogated by EGF, a Her-1 ligand and heregulin-β1, a Her-3 and Her-4 ligand. Most agents altered distribution throughout the phases of cell cycle to a certain degree, with the G1 phase accumulation observed for trastuzumab being potentiated in some combinations. Most of the agents, with the exception of doxorubicin and geldanamycin, did not promote apoptosis and appeared instead to be anti-proliferative. Geldanamycin had the greatest effect on Her-2 receptor density (approximately 80% by 24 hours) followed by EGF, heregulin and trastuzumab, with the biological molecules in combination with trastuzumab producing a further significant reduction. Conclusion: Endogenous Her-receptor ligands (EGF and heregulin) differentially altered the viability parameters for trastuzumab which could play a role in the emergence of clinical resistance to targeted therapy. Doxorubicin with concurrent trastuzumab significantly reduced cell viability compared to each single agent in both cell lines. Furthermore, the cytostatic and cytotoxic abilities of each of the other agents either mimicked trastuzumab alone or the selected agent alone when exposed concurrently.Dissertation (MSc)--University of Pretoria, 2013.gm2014PharmacologyUnrestricte

Proteomic assessment of potential in vitro hepatotoxicity models

Scientifically credible and valid biological systems are essential in pharmaceutical research and development. Standardizing in vitro preclinical hepatotoxicity is confounded by the diversity of origin of cells and the ability to retain hepatocellular functions. Key determinants of valid hepatotoxicity models are resemblance to primary human hepatocytes (PHHs), adaptability to high-throughput screening and biological applicability. Numerous in vitro models, including immortalized cell lines and hepatocyte-like cells (HLCs) derived from induced pluripotent stem cells (iPSCs), attempt to reflect features of PHH. Additional influencing factors are the mechanical and geometric environment which dictate functionality and suggest a role for spatial organization as a requirement for mimicking PHH. As there is poor correlation between the cellular genome and proteome, assessing the hepatic phenotypes using proteomics is essential to capture functional cellular responses. The aim of this research was to determine proteomic differences between PHHs and differentially cultured and sourced human hepatocyte-derived cell lines or differentiated HLCs. Additionally, hepatocyte models were used to generate non-specific, proteome-wide information associated with exposure to selected known hepatotoxins to identify potential proteomic signatures of hepatotoxicity.Thesis (PhD)--University of Pretoria, 2016.PharmacologyPhDUnrestricte

Proteomic responses of HepG2 cell monolayers and 3D spheroids to selected hepatotoxins

Despite the importance of hepatotoxicity testing in the development of new potential pharmaceuticals, standardized methods for preclinical in vitro hepatotoxicity is complicated by the perceived adequacy of approach, diversity of origin of cells, and the ability to retain a satisfactory hepatocellular phenotype. Additionally, the confidence with which cells mimic in vitro hepatocytes is dictated by the spatial dynamics of the cell culture microenvironment. This study sought to compare the proteome of conventional monolayer cultures of an immortalized hepatocyte cell line (HepG2) with more complex three-dimensional spheroid cultures to ascertain whether changes in culture technique better mimic the phenotype of hepatocytes and thereby improve responses to in vivo hepatotoxins. The proteome was assayed using isobaric tagging from six independent experiments, yielding relative quantitation of over 4600 proteins per multiplexed set. Approximately 34% of proteins present in all replicates differed between monolayer and 3D spheroid cultures. These data suggest that the cellular transition from an exponential to an equilibrium growth phase is inconsistent across biological replicates during spheroid formation which then variably alters the proteome from a stable phenotype in monolayers. Continuous exposure to hepatotoxins, did not implicate specific subsets of proteins in describing the associated mechanisms of toxicity of each drug. However, dynamic changes in HepG2 cells cultured as 3D spheroids were described. These data suggest that the duration of spheroid culture could be essential to reconcile the differences observed in the spheroid proteome to achieve reproducible proteomic transitions to a stable 3D spheroid phenotype.The National Research Foundation of South Africa (NRF) Thuthuka PhD funding track grant (No. 87880 ). TH was supported by a UK Commonwealth Split-site Scholarship ( ZACS-2014-653 ) and a Commonwealth, European and International Cambridge Trust Scholarship (USN: 302989247; App No: 10326363).https://www.elsevier.com/locate/toxlet2020-01-01hj2018Pharmacolog