Evaluation of drug effects regarding novel metal-based as well as natural anticancer compounds by means of bottom-up label-free LC-MS/MS-based proteome profiling

Für die Entwicklung eines Wirkstoffes und dessen klinische Anwendung ist ein besseres Verständnis seiner zellulären Targets und Wirkungsweise von enormer Bedeutung. Die derzeit eingesetzten phänotypischen Analysemethoden für Zellzyklus, Viabilität und Zelltod haben sich bewährt, liefern allerdings wenig Informationen über globale Effekte auf molekularer Ebene. Proteine werden zunehmend als Wirkstofftargets identifiziert und aufgrund ihrer Schlüsselfunktionen in zellulären Abläufen, weisen sie großes Potential für die Aufklärung von Wirkmechanismen auf. Deshalb wurde eine auf Flüssigchromatographie (LC) und Tandem-Massenspektrometrie (MS/MS) basierende Label-freie Proteomanalysemethode herangezogen, um Unterschiede in Proteinabundanzen zwischen mit Medikamenten behandelten und unbehandelten Zellen zu evaluieren. In verschiedenen Zellkuturmodellen wurden die Effekte von (i) den Metall-basierenden Antikrebswirkstoffen As¬¬2O3, KP46 (Gallium), KP772 (Lanthan), NKP-1339 (Ruthenium) und KP1537 (Platin) sowie (ii) dem natürlich vorkommenden Biphenol Kurkumin untersucht. Colonkarzinomzellen wurden mit den Metallverbindungen behandelt und anschließend in Zytoplasma und Krenextrakt fraktioniert, um einen tieferen Einblick in die stattfindenden regulatorischen Prozesse zu erhalten. Zur besseren Repräsentation der in vivo Situation, wurden Kurkumineffekte in Co-Kulturmodellen von Brustkrebszellen mit Krebsassoziierten-Fibroblasten untersucht. Hierfür wurden Gesamtzelllysate analysiert. Insgesamt wurden 5525 beziehungsweise 5780 Proteingruppen in den enzymatisch verdauten Colonkarzinom- und Brustkrebsproben mittels LC-MS/MS identifiziert. Die basalen Proteinkonzentrationen variierten stark zwischen den verschiedenen Zellen, auch wenn sie von dem gleichen Gewebe stammten, wodurch Proteinregulationen durch Wirkstoffbehandlungen entscheidend beeinflusst werden können. Die Untersuchungen der Effekte der Metallverbindungen auf das Proteom ermöglichten die Generierung von Hypothesen bezüglich ihrer Wirkmechanismen, die bis dahin unbekannt waren. Die Proteomanalysen der Kurkumineffekte zeigten, dass die Reaktion auf einen Wirkstoff stark kontextabhängig ist und die Tumormikroumgebung einen massiven Einfluss darauf ausübt. Die Ergebnisse dieser Dissertation könnten zu einer Verbesserung des Wirkstoffevaluierungsprozesses und von Patientenstratifizierungsstrategien führen.A better understanding of drug targets and the underlying modes of action are of great importance for drug development and application in clinical practice. The traditionally employed and well established phenotypic assays for cell cycle, viability and death analysis give little information about global effects on a molecular basis. Proteins are increasingly identified as drug targets and hold great potential for the elucidation of modes of action since they are the active players within cell, responsible for cellular fate and function. Therefore, a label-free quantification proteomics approach based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) was employed to evaluate protein abundances in treated and untreated cells, i.e. response profiling. Drug effects were investigated in different cell culture models by response profiling of (i) the metal-based anticancer drugs As¬¬2O3, KP46 (gallium), KP772 (lanthanum), NKP-1339 (ruthenium) and KP1537 (platinum) and (ii) the naturally occurring biphenol curcumin. Colon carcinoma cells were exposed to the metallodrugs and subsequently fractionated into cytoplasmic and nuclear extracts to gain a deeper insight into the regulatory effects. For a better representation of the in vivo situation, investigations on curcumin effects executed not only with single cell type cultures but also with tumor-stroma co-culture models of breast cancer and cancer-associated fibroblast-like cells. Whole cell lysates were obtained for this study. Overall, 5525 and 5780 protein groups were identified by LC-MS/MS analysis of in-solution digested colon and breast cancer samples, respectively. Remarkably great differences of basal protein abundances were observed in the diverse cells, even if they derived from the same tissue. This circumstance may substantially affect protein regulations upon drug exposure. Global effects of the investigated metallodrugs on the proteome were characterized and hypotheses on hitherto unrecognized mechanisms were generated. Response profiling of curcumin demonstrated strong context-dependent effects, emphasizing the massive contribution of the microenvironment to drug responses. The findings of this thesis might contribute to the improvement of drug evaluation as well as patient stratification strategies

Quantification of Cytokines secreted by primary human cells using multiple reaction monitoring: evaluation of analytical parameters

Determination of secreted proteins provides highly valuable information about cell functions. While the typical methods for the determination of biologically relevant but low-abundant molecular species still relies on the use of specific antibodies, mass spectrometry-based methods are now gaining sufficient sensitivity to cope with such challenges as well. In the current study we have identified several cytokines and chemokines which were induced in primary human umbilical vein endothelial cells upon inflammatory activation. Based on the high-resolution mass spectrometry data obtained with a Q Exactive orbitrap, we built an MRM method to quantify the most relevant molecules selected from the screening experiment. All experimental data are available via ProteomeXchange, PXD002211/12, and Panorama, www.panoramaweb.org. Using nano-flow Chip-HPLC coupled to a 6490 triple-quadrupole MS for MRM analyses we achieved calibration curves covering a linear range of four orders of magnitude and detection limits in the low attomol per microliter concentration range. Carryover was consistently less than 0.005%, the accuracy was between 80% and 120%, and the median coefficient of variation for LC/MS was only 2.2%. When including the variance of quantification introduced by cell culture and digestion, the coefficient of variation was less than 20% for most peptides. With appropriate marker molecules we monitored typical variations introduced by cell culture caused by differences in cell numbers, proliferative states and cell death. As a result, here, we present a robust and efficient MRM-based assay for the accurate and sensitive determination of cytokines and chemokines representative for functional cell states and including comprehensive quality controls

Comprehensive Assessment of Proteins Regulated by Dexamethasone Reveals Novel Effects in Primary Human Peripheral Blood Mononuclear Cells

Inflammation is a physiological process involved in many diseases. Monitoring proteins involved in regulatory effects may help to improve our understanding of inflammation. We have analyzed proteome alterations induced in peripheral blood mononuclear cells (PBMCs) upon inflammatory activation in great detail using high-resolution mass spectrometry. Moreover, the activated cells were treated with dexamethasone to investigate their response to this antiphlogistic drug. From a total of 6886 identified proteins, 469 proteins were significantly regulated upon inflammatory activation. Data are available via ProteomeXchange with identifiers PXD001415–23. Most of these proteins were counter-regulated by dexamethasone, with some exceptions concerning members of the interferon-induced protein family. To confirm some of these results, we performed targeted MRM analyses of selected peptides. The inflammation-induced upregulation of proteins such as IL-1β, IL-6, CXCL2, and GROα was confirmed, however, with strong quantitative interindividual differences. Furthermore, the inability of dexamethasone to downregulate inflammation-induced proteins such as PTX3 and TSG6 was clearly demonstrated. In conclusion, the relation of cell function as well as drug-induced modulation thereof was successfully mapped to proteomes, suggesting targeted analysis as a novel and powerful drug evaluation method. Although most consequences of dexamethasone were found to be compatible with the expected mode of action, some unexpected but significant observations may be related to adverse effects

Coffee consumption modulates inflammatory processes in an individual fashion

Anti-inflammatory effects of coffee consumption have been reported to be caused by caffeine and adenosine receptor signaling. However, contradictory effects have been observed. Many kinds of chronic diseases are linked to inflammation; therefore a profound understanding of potential effects of coffee consumption is desirable. We performed ex vivo experiments with eight individuals investigating peripheral blood mononuclear cells isolated from venous blood before and after coffee consumption, as well as in vitro experiments applying caffeine on isolated cells. After in vitro inflammatory stimulation of the cells, released cytokines, chemokines and eicosanoids were determined and quantified using targeted mass spectrometric methods. Remarkably, the release of inflammation mediators IL6, IL8, GROA, CXCL2, CXCL5 as well as PGA2, PGD2, PGE2, LTC4, LTE4 and 15S-HETE was significantly affected after coffee consumption. While in several individuals coffee consumption or caffeine treatment caused significant down-regulation of most inflammation mediators, in other healthy individuals exactly the opposite effects were observed. Ruling out age, sex, coffee consumption habits, the metabolic kinetics of caffeine in blood and the individual amount of regulatory T-cells or CD39 expression as predictive parameters, we demonstrated here that coffee consumption may have significant pro- or anti-inflammatory effects in an individual fashion

Metabolic, Anti-apoptotic and Immune Evasion Strategies of Primary Human Myeloma Cells Indicate Adaptations to Hypoxia*

Multiple Myeloma (MM) is an incurable plasma cell malignancy primarily localized within the bone marrow (BM). It develops from a premalignant stage, monoclonal gammopathy of undetermined significance (MGUS), often via an intermediate stage, smoldering MM (SMM). The mechanisms of MM progression have not yet been fully understood, all the more because patients with MGUS and SMM already carry similar initial mutations as found in MM cells. Over the last years, increased importance has been attributed to the tumor microenvironment and its role in the pathophysiology of the disease. Adaptations of MM cells to hypoxic conditions in the BM have been shown to contribute significantly to MM progression, independently from the genetic predispositions of the tumor cells. Searching for consequences of hypoxia-induced adaptations in primary human MM cells, CD138-positive plasma cells freshly isolated from BM of patients with different disease stages, comprising MGUS, SMM, and MM, were analyzed by proteome profiling, which resulted in the identification of 6218 proteins. Results have been made fully accessible via ProteomeXchange with identifier PXD010600. Data previously obtained from normal primary B cells were included for comparative purposes. A principle component analysis revealed three clusters, differentiating B cells as well as MM cells corresponding to less and more advanced disease stages. Comparing these three clusters pointed to the alteration of pathways indicating adaptations to hypoxic stress in MM cells on disease progression. Protein regulations indicating immune evasion strategies ofMMcells were determined, supported by immunohistochemical staining, as well as transcription factors involved in MM development and progression. Protein regulatory networks related to metabolic adaptations of the cells became apparent. Results were strengthened by targeted analyses of a selected panel of metabolites in MM cells and MM-associated fibroblasts. Based on our data, new opportunities may arise for developing therapeutic strategies targeting myeloma disease progression. Molecular & Cellular Proteomics 18: 936-953, 2019. DOI:10.1074/mcp.RA119.001390

Adherence to treatment of osteoporosis: a need for study

Adherence to anti-osteoporosis medications is currently low and is associated with poor anti-fracture efficacy. This manuscript reviews the potential design of clinical studies that aim to demonstrate improved adherence, with new chemical entities to be used in the management of osteoporosis. Introduction Several medications have been unequivocally shown to decrease fracture rates in clinical trials. However, in real life settings, long-term persistence and compliance to anti-osteoporosis medication is poor, hence decreasing the clinical benefits for patients. Methods An extensive search of Medline from 1985 to 2006 retrieved all trials including the keywords osteoporosis, compliance, persistence or adherence followed by a critical appraisal of the data obtained through a consensus expert meeting. Results The impact of non-adherence on the clinical development of interventions is reviewed, so that clinicians, regulatory agencies and reimbursement agencies might be better informed of the problem, in order to stimulate the necessary research to document adherence. Conclusion Adherence to therapy is a major problem in the treatment of osteoporosis. Both patients and medication factors are involved. Adherence studies are an important aspect of outcomes studies, but study methodologies are not well developed at the moment and should be improved. Performing adherence studies will be stimulated when registration authorities accept the result of these studies and include the relevant information in Sect. 5.1 of the summary of product characteristics. Reimbursement authorities might also consider such studies as important information for decisions on reimbursement

Proteome Profiling of Breast Cancer Biopsies Reveals a Wound Healing Signature of Cancer-Associated Fibroblasts

Breast cancer is still the most common type of cancer in women; an important role in carcinogenesis is actually attributed to cancer-associated fibroblasts. In this study, we investigated whether it is possible to assess the functional state of cancer-associated fibroblasts through tumor tissue proteome profiling. Tissue proteomics was performed on tumor-central, tumor-near, and tumor-distant biopsy sections from breast adenocarcinoma patients, which allowed us to identify 2074 proteins. Data were interpreted referring to reference proteome profiles generated from primary human mammary fibroblasts comprising 4095 proteins. These cells were analyzed in quiescent cell state as well as after in vitro treatment with TGFβ or IL-1β, stimulating wound healing or inflammatory processes, respectively. Representative for cancer cells, we investigated the mammary carcinoma cell line ZR-75-1, identifying 5212 proteins. All mass analysis data have been made fully accessible via ProteomeXchange, DOI PXD001311 and PXD001323-8. Comparison of tissue proteomics data with all of those reference profiles revealed predominance of cancer cell-derived proteins within the tumor and fibroblast-derived proteins in the tumor-distant tissue sections. Remarkably, proteins characteristic for acute inflammation were hardly identified in the tissue samples. In contrast, several proteins found by us to be induced by TGFβ in mammary fibroblasts, including fibulin-5, SLC2A1, and MUC18, were positively identified in all tissue samples, with relatively higher abundance in tumor neighboring tissue sections. These findings indicate a predominance of cancer-associated fibroblasts with wound healing activities localized around tumors

Regulation of selected metabolites assessed by the AbsoluteIDQ p180 metabolomics kit illustrating the contrasting inflammation modulating effects of dexamethasone (dex) and M rac.

<p>Again, HUVECs were stimulated with IL-1βeta (IL-1β) and additionally treated with dexamethasone or M rac extract (M rac). Error bars are derived from two technical and two biological replicates. (p-values: *, p<0.05 and **, p<0.005.)</p