Decitabine-induced DNA methylation-mediated transcriptomic reprogramming in human breast cancer cell lines; the impact of DCK overexpression

Decitabine (DAC), a DNA methyltransferase (DNMT) inhibitor, is tested in combination with conventional anticancer drugs as a treatment option for various solid tumors. Although epigenome modulation provides a promising avenue in treating resistant cancer types, more studies are required to evaluate its safety and ability to normalize the aberrant transcriptional profiles. As deoxycytidine kinase (DCK)-mediated phosphorylation is a rate-limiting step in DAC metabolic activation, we hypothesized that its intracellular overexpression could potentiate DAC’s effect on cell methylome and thus increase its therapeutic efficacy. Therefore, two breast cancer cell lines, JIMT-1 and T-47D, differing in their molecular characteristics, were transfected with a DCK expression vector and exposed to low-dose DAC (approximately IC20). Although transfection resulted in a significant DCK expression increase, further enhanced by DAC exposure, no transfection-induced changes were found at the global DNA methylation level or in cell viability. In parallel, an integrative approach was applied to decipher DAC-induced, methylation-mediated, transcriptomic reprogramming. Besides large-scale hypomethylation, accompanied by up-regulation of gene expression across the entire genome, DAC also induced hypermethylation and down-regulation of numerous genes in both cell lines. Interestingly, TET1 and TET2 expression halved in JIMT-1 cells after DAC exposure, while DNMTs’ changes were not significant. The protein digestion and absorption pathway, containing numerous collagen and solute carrier genes, ranking second among membrane transport proteins, was the top enriched pathway in both cell lines when hypomethylated and up-regulated genes were considered. Moreover, the calcium signaling pathway, playing a significant role in drug resistance, was among the top enriched in JIMT-1 cells. Although low-dose DAC demonstrated its ability to normalize the expression of tumor suppressors, several oncogenes were also up-regulated, a finding, that supports previously raised concerns regarding its broad reprogramming potential. Importantly, our research provides evidence about the involvement of active demethylation in DAC-mediated transcriptional reprogramming.publishedVersio

Decitabine potentiates efficacy of doxorubicin in a preclinical trastuzumab-resistant HER2-positive breast cancer models

Acquired drug resistance and metastasis in breast cancer (BC) are coupled with epigenetic deregulation of gene expression. Epigenetic drugs, aiming to reverse these aberrant transcriptional patterns and sensitize cancer cells to other therapies, provide a new treatment strategy for drug-resistant tumors. Here we investigated the ability of DNA methyltransferase (DNMT) inhibitor decitabine (DAC) to increase the sensitivity of BC cells to anthracycline antibiotic doxorubicin (DOX). Three cell lines representing different molecular BC subtypes, JIMT-1, MDA-MB-231 and T-47D, were used to evaluate the synergy of sequential DAC + DOX treatment in vitro. The cytotoxicity, genotoxicity, apoptosis, and migration capacity were tested in 2D and 3D cultures. Moreover, genome-wide DNA methylation and transcriptomic analyses were employed to understand the differences underlying DAC responsiveness. The ability of DAC to sensitize trastuzumab-resistant HER2-positive JIMT-1 cells to DOX was examined in vivo in an orthotopic xenograft mouse model. DAC and DOX synergistic effect was identified in all tested cell lines, with JIMT-1 cells being most sensitive to DAC. Based on the whole-genome data, we assume that the aggressive behavior of JIMT-1 cells can be related to the enrichment of epithelial-to-mesenchymal transition and stemness-associated pathways in this cell line. The four-week DAC + DOX sequential administration significantly reduced the tumor growth, DNMT1 expression, and global DNA methylation in xenograft tissues. The efficacy of combination therapy was comparable to effect of pegylated liposomal DOX, used exclusively for the treatment of metastatic BC. This work demonstrates the potential of epigenetic drugs to modulate cancer cells' sensitivity to other forms of anticancer therapy.publishedVersio

The alamar blue assay in the context of safety testing of nanomaterials

The Alamar Blue (AB) assay is widely used to investigate cytotoxicity, cell proliferation and cellular metabolic activity within different fields of toxicology. The use of the assay with nanomaterials (NMs) entails specific aspects including the potential interference of NMs with the test. The procedure of the AB assay applied for testing NMs is described in detail and step-by-step, from NM preparation, cell exposure, inclusion of interference controls, to the analysis and interpretation of the results. Provided that the proper procedure is followed, and relevant controls are included, the AB assay is a reliable and high throughput test to evaluate the cytotoxicity/proliferation/metabolic response of cells exposed to NMs

The colony forming efficiency assay for toxicity testing of nanomaterials—Modifications for higher-throughput

To cope with the high number of nanomaterials manufactured, it is essential to develop high-throughput methods for in vitro toxicity screening. At the same time, the issue with interference of the nanomaterial (NM) with the read-out or the reagent of the assay needs to be addressed to avoid biased results. Thus, validated label-free methods are urgently needed for hazard identification of NMs to avoid unintended adverse effects on human health. The colony forming efficiency (CFE) assay is a label- and interference-free method for quantification of cytotoxicity by cell survival and colony forming efficiency by CFE formation. The CFE has shown to be compatible with toxicity testing of NMs. Here we present an optimized protocol for a higher-throughput set up

The colony forming efficiency assay for toxicity testing of nanomaterials—Modifications for higher-throughput

To cope with the high number of nanomaterials manufactured, it is essential to develop high-throughput methods for in vitro toxicity screening. At the same time, the issue with interference of the nanomaterial (NM) with the read-out or the reagent of the assay needs to be addressed to avoid biased results. Thus, validated label-free methods are urgently needed for hazard identification of NMs to avoid unintended adverse effects on human health. The colony forming efficiency (CFE) assay is a label- and interference-free method for quantification of cytotoxicity by cell survival and colony forming efficiency by CFE formation. The CFE has shown to be compatible with toxicity testing of NMs. Here we present an optimized protocol for a higher-throughput set up

The colony forming efficiency assay for toxicity testing of nanomaterials – Modifications for higher-throughput

To cope with the high number of nanomaterials manufactured, it is essential to develop high-throughput methods for in vitro toxicity screening. At the same time, the issue with interference of the nanomaterial (NM) with the read-out or the reagent of the assay needs to be addressed to avoid biased results. Thus, validated label-free methods are urgently needed for hazard identification of NMs to avoid unintended adverse effects on human health. The colony forming efficiency (CFE) assay is a label- and interference-free method for quantification of cytotoxicity by cell survival and colony forming efficiency by CFE formation. The CFE has shown to be compatible with toxicity testing of NMs. Here we present an optimized protocol for a higher-throughput set up

Advanced Respiratory Models for Hazard Assessment of Nanomaterials. Performance of Mono-, Co- and Tricultures

Advanced in vitro models are needed to support next-generation risk assessment (NGRA), moving from hazard assessment based mainly on animal studies to the application of new alternative methods (NAMs). Advanced models must be tested for hazard assessment of nanomaterials (NMs). The aim of this study was to perform an interlaboratory trial across two laboratories to test the robustness of and optimize a 3D lung model of human epithelial A549 cells cultivated at the air–liquid interface (ALI). Potential change in sensitivity in hazard identification when adding complexity, going from monocultures to co- and tricultures, was tested by including human endothelial cells EA.hy926 and differentiated monocytes dTHP-1. All models were exposed to NM-300K in an aerosol exposure system (VITROCELL® cloud-chamber). Cyto- and genotoxicity were measured by AlamarBlue and comet assay. Cellular uptake was investigated with transmission electron microscopy. The models were characterized by confocal microscopy and barrier function tested. We demonstrated that this advanced lung model is applicable for hazard assessment of NMs. The results point to a change in sensitivity of the model by adding complexity and to the importance of detailed protocols for robustness and reproducibility of advanced in vitro model

Advanced Respiratory Models for Hazard Assessment of Nanomaterials. Performance of Mono-, Co- and Tricultures

Advanced in vitro models are needed to support next-generation risk assessment (NGRA), moving from hazard assessment based mainly on animal studies to the application of new alternative methods (NAMs). Advanced models must be tested for hazard assessment of nanomaterials (NMs). The aim of this study was to perform an interlaboratory trial across two laboratories to test the robustness of and optimize a 3D lung model of human epithelial A549 cells cultivated at the air–liquid interface (ALI). Potential change in sensitivity in hazard identification when adding complexity, going from monocultures to co- and tricultures, was tested by including human endothelial cells EA.hy926 and differentiated monocytes dTHP-1. All models were exposed to NM-300K in an aerosol exposure system (VITROCELL® cloud-chamber). Cyto- and genotoxicity were measured by AlamarBlue and comet assay. Cellular uptake was investigated with transmission electron microscopy. The models were characterized by confocal microscopy and barrier function tested. We demonstrated that this advanced lung model is applicable for hazard assessment of NMs. The results point to a change in sensitivity of the model by adding complexity and to the importance of detailed protocols for robustness and reproducibility of advanced in vitro model

Advanced Respiratory Models for Hazard Assessment of Nanomaterials. Performance of Mono-, Co- and Tricultures

Advanced in vitro models are needed to support next-generation risk assessment (NGRA), moving from hazard assessment based mainly on animal studies to the application of new alternative methods (NAMs). Advanced models must be tested for hazard assessment of nanomaterials (NMs). The aim of this study was to perform an interlaboratory trial across two laboratories to test the robustness of and optimize a 3D lung model of human epithelial A549 cells cultivated at the air–liquid interface (ALI). Potential change in sensitivity in hazard identification when adding complexity, going from monocultures to co- and tricultures, was tested by including human endothelial cells EA.hy926 and differentiated monocytes dTHP-1. All models were exposed to NM-300K in an aerosol exposure system (VITROCELL® cloud-chamber). Cyto- and genotoxicity were measured by AlamarBlue and comet assay. Cellular uptake was investigated with transmission electron microscopy. The models were characterized by confocal microscopy and barrier function tested. We demonstrated that this advanced lung model is applicable for hazard assessment of NMs. The results point to a change in sensitivity of the model by adding complexity and to the importance of detailed protocols for robustness and reproducibility of advanced in vitro model

Advanced Respiratory Models for Hazard Assessment of Nanomaterials. Performance of Mono-, Co- and Tricultures

Advanced in vitro models are needed to support next-generation risk assessment (NGRA), moving from hazard assessment based mainly on animal studies to the application of new alternative methods (NAMs). Advanced models must be tested for hazard assessment of nanomaterials (NMs). The aim of this study was to perform an interlaboratory trial across two laboratories to test the robustness of and optimize a 3D lung model of human epithelial A549 cells cultivated at the air–liquid interface (ALI). Potential change in sensitivity in hazard identification when adding complexity, going from monocultures to co- and tricultures, was tested by including human endothelial cells EA.hy926 and differentiated monocytes dTHP-1. All models were exposed to NM-300K in an aerosol exposure system (VITROCELL® cloud-chamber). Cyto- and genotoxicity were measured by AlamarBlue and comet assay. Cellular uptake was investigated with transmission electron microscopy. The models were characterized by confocal microscopy and barrier function tested. We demonstrated that this advanced lung model is applicable for hazard assessment of NMs. The results point to a change in sensitivity of the model by adding complexity and to the importance of detailed protocols for robustness and reproducibility of advanced in vitro model