| 1. |
- Flörkemeier, Inken, et al.
(författare)
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Newly developed dual topoisomerase inhibitor P8-D6 is highly active in ovarian cancer
- 2021
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Ingår i: THERAPEUTIC ADVANCES IN MEDICAL ONCOLOGY. - : Sage Publications. - 1758-8340 .- 1758-8359. ; 13
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Tidskriftsartikel (refereegranskat)abstract
- Background: Ovarian cancer (OvCa) constitutes a rare and highly aggressive malignancy and is one of the most lethal of all gynaecologic neoplasms. Due to chemotherapy resistance and treatment limitations because of side effects, OvCa is still not sufficiently treatable. Hence, new drugs for OvCa therapy such as P8-D6 with promising antitumour properties have a high clinical need. The benzo[c]phenanthridine P8-D6 is an effective inductor of apoptosis by acting as a dual topoisomerase I/II inhibitor.Methods: In the present study, the effectiveness of P8-D6 on OvCa was investigated in vitro. In various OvCa cell lines and ex vivo primary cells, the apoptosis induction compared with standard therapeutic agents was determined in two-dimensional monolayers. Expanded by three-dimensional and co-culture, the P8-D6 treated cells were examined for changes in cytotoxicity, apoptosis rate and membrane integrity via scanning electron microscopy (SEM). Likewise, the effects of P8-D6 on non-cancer human ovarian surface epithelial cells and primary human hepatocytes were determined.Results: This study shows a significant P8-D6-induced increase in apoptosis and cytotoxicity in OvCa cells which surpasses the efficacy of well-established drugs like cisplatin or the topoisomerase inhibitors etoposide and topotecan. Non-cancer cells were affected only slightly by P8-D6. Moreover, no hepatotoxic effect in in vitro studies was detected.Conclusion: P8-D6 is a strong and rapid inductor of apoptosis and might be a novel treatment option for OvCa therapy.
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| 2. |
- Handin, Niklas, et al.
(författare)
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Conditions for maintenance of hepatocyte differentiation and function in 3D cultures
- 2021
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Ingår i: iScience. - : Cell Press. - 2589-0042. ; 24:11
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Tidskriftsartikel (refereegranskat)abstract
- Spheroid cultures of primary human hepatocytes (PHH) are used in studies of hepatic drug metabolism and toxicity. The cultures are maintained under different cone-lions, with possible confounding results. We performed an in-depth analysis of the influence of various culture conditions to find the optimal conditions for the maintenance of an in vivo like phenotype. The formation, protein expression, and function of PHH spheroids were followed for three weeks in a high-throughput 384-well format. Medium composition affected spheroid histology, global proteome profile, drug metabolism and drug-induced toxicity. No epithelial-mesenchymel transition was observed. Media with fasting glucose and insulin levels gave spheroids with phenotypes closest to normal PHH. The most expensive medium resulted in PHH features most divergent from that of native PHH. Our results provide a protocol for culture of healthy PHH with maintained function a prerequisite for studies of hepatocyte homeostasis and more reproducible hepatocyte research.
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| 6. |
- Wegler, Christine, et al.
(författare)
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Global variability analysis of mRNA and protein concentrations across and within human tissues
- 2020
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Ingår i: NAR Genomics and Bioinformatics. - : Oxford University Press (OUP). - 2631-9268. ; 2:1
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Tidskriftsartikel (refereegranskat)abstract
- Genes and proteins show variable expression patterns throughout the human body. However, it is not clear whether relative differences in mRNA concentrations are retained on the protein level. Furthermore, inter-individual protein concentration variability within single tissue types has not been comprehensively explored. Here, we used the Gini index for in-depth concentration variability analysis of publicly available transcriptomics and proteomics data, and of an in-house proteomics dataset of human liver and jejunum from 38 donors. We found that the transfer of concentration variability from mRNA to protein is limited, that established ‘reference genes’ for data normalization vary markedly at the protein level, that protein concentrations cover a wide variability spectrum within single tissue types, and that concentration variability analysis can be a convenient starting point for identifying disease-associated proteins and novel biomarkers. Our results emphasize the importance of considering individual concentration levels, as opposed to population averages, for personalized systems biology analysis.
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| 7. |
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| 8. |
- Ölander, Magnus, et al.
(författare)
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A simple approach for restoration of differentiation and function in cryopreserved human hepatocytes
- 2019
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Ingår i: Archives of Toxicology. - : Springer Science and Business Media LLC. - 0340-5761 .- 1432-0738. ; 93:3, s. 819-829
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Tidskriftsartikel (refereegranskat)abstract
- Primary human hepatocytes are used in all facets of liver research, from in vitro studies of xenobiotic disposition and toxicity to the clinical management of liver failure. Unfortunately, cellular stress during isolation and cryopreservation causes a highly unpredictable loss of the ability to attach and form cell-matrix and cell-cell interactions. Reasoning that this problem could be mitigated at the post-thawing stage, we applied label-free quantitative global proteomics to analyze differences between attached and non-attached fractions of cryopreserved human hepatocyte batches. Hepatocytes that were unable to attach to a collagen matrix showed many signs of cellular stress, including a glycolytic phenotype and activation of the heat shock response, ultimately leading to apoptosis activation. Further analysis of the activated stress pathways revealed an increase in early apoptosis immediately post-thawing, which suggested the possibility of stress reversal. Therefore, we transiently treated the cells with compounds aimed at decreasing cellular stress via different mechanisms. Brief exposure to the pan-caspase apoptosis inhibitor Z-VAD-FMK restored attachment ability and promoted a differentiated morphology, as well as formation of 3D spheroids. Further, Z-VAD-FMK treatment restored metabolic and transport functions, with maintained sensitivity to hepatotoxic insults. Altogether, this study shows that differentiation and function of suboptimal human hepatocytes can be restored after cryopreservation, thus markedly increasing the availability of these precious cells.
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| 9. |
- Ölander, Magnus, et al.
(författare)
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Apoptosis detection by quantification of cell debris in bright-field microscopy images
- 2022
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Ingår i: Apoptosis and cancer. - New York : Humana Press. - 9781071625521 - 9781071625538 ; , s. 27-33
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Bokkapitel (refereegranskat)abstract
- We describe a simple protocol for quantification of apoptosis by counting subcellular debris particles in bright-field microscopy images of cell culture media samples. The only necessary equipment is a bright-field microscope (fitted with a digital camera) and a computer for image processing and analysis. The method gives comparable results to established fluorescence markers in several different applications and is, in principle, compatible with any culture format. Given its simplicity, accessibility, and inexpensive nature, the method can complement or provide an alternative to other methods for apoptosis detection.
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| 11. |
- Ölander, Magnus, et al.
(författare)
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Cell-type-resolved proteomic analysis of the human liver
- 2020
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Ingår i: Liver international. - : John Wiley & Sons. - 1478-3223 .- 1478-3231. ; 40:7, s. 1770-1780
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Tidskriftsartikel (refereegranskat)abstract
- Background & Aims The human liver functions through a complex interplay between parenchymal and non-parenchymal cells. Mass spectrometry-based proteomic analysis of intact tissue has provided an in-depth view of the human liver proteome. However, the predominance of parenchymal cells (hepatocytes) means that the total tissue proteome mainly reflects hepatocyte expression. Here we therefore set out to analyse the proteomes of the major parenchymal and non-parenchymal cell types in the human liver.Methods We applied quantitative label-free proteomic analysis on the major cell types of the human liver: hepatocytes, liver endothelial cells, Kupffer cells and hepatic stellate cells.Results We identified 9791 proteins, revealing distinct protein expression profiles across cell types, whose in vivo relevance was shown by the presence of cell-type-specific proteins. Analysis of proteins related to the immune system indicated that mechanisms of immune-mediated liver injury include the involvement of several cell types. Furthermore, in-depth investigation of proteins related to the absorption, distribution, metabolism, excretion and toxicity (ADMET) of xenobiotics showed that ADMET-related tasks are not exclusively confined to hepatocytes, and that non-parenchymal cells may contribute to drug transport and metabolism.Conclusions Overall, the data we provide constitute a unique resource for exploring the proteomes of the major types of human liver cells, which will facilitate an improved understanding of the human liver in health and disease.
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| 12. |
- Ölander, Magnus, et al.
(författare)
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Hepatocyte size fractionation allows dissection of human liver zonation
- 2021
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Ingår i: Journal of Cellular Physiology. - : John Wiley & Sons. - 0021-9541 .- 1097-4652. ; 236:8, s. 5885-5894
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Tidskriftsartikel (refereegranskat)abstract
- Human hepatocytes show marked differences in cell size, gene expression, and function throughout the liver lobules, an arrangement termed liver zonation. However, it is not clear if these zonal size differences, and the associated phenotypic differences, are retained in isolated human hepatocytes, the “gold standard” for in vitro studies of human liver function. Here, we therefore explored size differences among isolated human hepatocytes and investigated whether separation by size can be used to study liver zonation in vitro. We used counterflow centrifugal elutriation to separate cells into different size fractions and analyzed them with label-free quantitative proteomics, which revealed an enrichment of 151 and 758 proteins (out of 5163) in small and large hepatocytes, respectively. Further analysis showed that protein abundances in different hepatocyte size fractions recapitulated the in vivo expression patterns of previously described zonal markers and biological processes. We also found that the expression of zone-specific cytochrome P450 enzymes correlated with their metabolic activity in the different fractions. In summary, our results show that differences in hepatocyte size matches zonal expression patterns, and that our size fractionation approach can be used to study zone-specific liver functions in vitro.
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| 13. |
- Ölander, Magnus, et al.
(författare)
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Image-based quantification of cell debris as a measure of apoptosis
- 2019
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 91:9, s. 5548-5552
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Tidskriftsartikel (refereegranskat)abstract
- Apoptosis is a controlled form of cell death that can be induced by various diseases and exogenous toxicants. Common apoptosis detection methods rely on fluorescent markers, which necessitates the use of costly reagents and time-consuming labeling procedures. Label-free methods avoid these problems, but often require specialized instruments instead. Here, we utilize apoptotic cell disintegration to develop a novel label-free detection method based on the quantification of subcellular debris particles in bright-field microscopy images. Debris counts show strong correlations with fluorescence-based annexin V staining, and can be used to study concentration-dependent and temporal apoptosis activation. The method is rapid, low-cost, and easy to apply, as the only experimental step comprises bright-field imaging of culture media samples, followed by automated image processing. The late-stage nature of the debris measurement means that the method can complement other, established apoptosis assays, and its accessibility will allow a wider community of researchers to study apoptotic cell death.
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| 14. |
- Ölander, Magnus
(författare)
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Proteomic and Functional Analysis of In Vitro Systems for Studies of Drug Disposition in the Human Small Intestine and Liver
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- To reach the bloodstream, an orally administered drug must be absorbed through the small intestine and avoid extensive clearance in the liver. Estimating these parameters in vitro is therefore important in drug discovery and development. This can be achieved with cellular models that simulate human organ function, such as Caco-2 cells and primary hepatocytes. No model fits every scenario, however, and this thesis aimed at using proteomic and functional analysis to better understand and increase the applicability of in vitro models based on Caco-2 cells and human hepatocytes.First, the proteome of filter-grown Caco-2 cells was analyzed. This included near-complete coverage of enterocyte-related proteins, and over 300 ADME proteins. Further, by scaling uptake transport kinetics from Caco-2 cells to human jejunum, the importance of considering in vitro-in vivo expression differences to correctly interpret in vitro transport studies was demonstrated.Focus was then turned to hepatocytes, where proteomics was used as a basis for the successful development of an apoptosis inhibition protocol for restoration of attachment properties and functionality in suboptimal batches of cryopreserved human hepatocytes. As a spin-off project, image-based quantification of cell debris was developed into a novel apoptosis detection method.Next, the in vivo heterogeneity of human hepatocytes was explored in an in vitro setting, where it was observed that human hepatocyte batches contain a wide range of cell sizes. By separating the cells into different size fractions, it was found that hepatocyte size corresponds to the microarchitectural zone of origin in the liver. Size separation can thus be used to study zonated liver functions in vitro.Finally, the proteomes of the major types of non-parenchymal liver cells were analyzed, i.e. liver sinusoidal endothelial cells, Kupffer cells, and hepatic stellate cells. The different cell types all had distinctly different proteomes, and the expression of certain important ADME proteins indicated that non-parenchymal cells participate in drug disposition.In conclusion, this thesis has improved the phenotypic understanding and extended the applicability of Caco-2 cells and primary human hepatocytes, two of the most important in vitro models for studies of small intestinal and hepatic drug disposition.
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| 15. |
- Ölander, Magnus, et al.
(författare)
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The Proteome of Filter-Grown Caco-2 Cells With a Focus on Proteins Involved in Drug Disposition
- 2016
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Ingår i: Journal of Pharmaceutical Sciences. - : Elsevier BV. - 0022-3549 .- 1520-6017. ; 105:2, s. 817-827
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Tidskriftsartikel (refereegranskat)abstract
- Caco-2 cells are widely used in studies of intestinal cell physiology and drug transport. Here, the global proteome of filter-grown Caco-2 cells was quantified using the total protein approach and compared with the human colon and jejunum proteomes. In total, 8096 proteins were identified. In-depth analysis of proteins defining enterocyte differentiation—including brush-border hydrolases, integrins, and adherens and tight junctions—gave near-complete coverage of the expected proteins. Three hundred twenty-seven absorption, distribution, metabolism and excretion proteins were identified, including 112 solute carriers and 20 ATP-binding cassette transporters. OATP2B1 levels were 16-fold higher in Caco-2 cells than in jejunum. To investigate the impact of this difference on in vitro-in vivo extrapolations, we studied the uptake kinetics of the OATP2B1 substrate pitavastatin in Caco-2 monolayers, and found that the contribution of OATP2B1 was 60%-70% at clinically relevant intestinal concentrations. Pitavastatin kinetics was combined with transporter concentrations to model the contribution of active transport and membrane permeation in the jejunum. The lower OATP2B1 expression in jejunum led to a considerably lower transporter contribution (<5%), suggesting that transmembrane diffusion dominates pitavastatin absorption in vivo. In conclusion, we present the first in-depth quantification of the filter-grown Caco-2 proteome. We also demonstrate the crucial importance of considering transporter expression levels for correct interpretation of drug transport routes across the human intestine.
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