Cancer cell resistance to therapeutics can result from acquired or de novo-mediated factors. Here, we have utilised advanced breast cancer cell culture models to elucidate de novo doxorubicin ...resistance mechanisms.
The response of breast cancer cell lines (MCF-7 and MDA-MB-231) to doxorubicin was examined in an in vitro three-dimensional (3D) cell culture model. Cells were cultured with Matrigel™ enabling cellular arrangements into a 3D architecture in conjunction with cell-to-extracellular matrix (ECM) contact.
Breast cancer cells cultured in a 3D ECM-based model demonstrated altered sensitivity to doxorubicin, when compared to those grown in corresponding two-dimensional (2D) monolayer culture conditions. Investigations into the factors triggering the observed doxorubicin resistance revealed that cell-to-ECM interactions played a pivotal role. This finding correlated with the up-regulation of pro-survival proteins in 3D ECM-containing cell culture conditions following exposure to doxorubicin. Inhibition of integrin signalling in combination with doxorubicin significantly reduced breast cancer cell viability. Furthermore, breast cancer cells grown in a 3D ECM-based model demonstrated a significantly reduced proliferation rate in comparison to cells cultured in 2D conditions.
Collectively, these novel findings reveal resistance mechanisms which may contribute to reduced doxorubicin sensitivity.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Plasmodium falciparum gametocytes, specifically mature stages, are the only stage in man transmissible to the mosquito vector responsible for malaria transmission. Anti-malarial drugs capable of ...killing these forms are considered essential for the eradication of malaria. The comprehensive profiling of in vitro activity of anti-malarial compounds against both early (I-III) and late (IV-V) stage P. falciparum gametocytes, along with the high throughput screening (HTS) outcomes from the MMV malaria box are described.
Two anti-gametocyte HTS assays based on confocal fluorescence microscopy, utilizing both a gametocyte specific protein (pfs16-Luc-GFP) and a viability marker (MitoTracker Red CM-H2XRos) (MTR), were used for the measurement of anti-gametocytocidal activity. This combination provided a direct observation of gametocyte number per assay well, whilst defining the viability of each gametocyte imaged.
IC50 values were obtained for 36 current anti-malarial compounds for activities against asexual, early and late stage gametocytes. The MMV malaria box was screened and actives progressed for IC50 evaluation. Seven % of the "drug-like" and 21% of the "probe-like" compounds from the MMV malaria box demonstrated equivalent activity against both asexual and late stage gametocytes.
The assays described were shown to selectively identify compounds with gametocytocidal activity and have been demonstrated suitable for HTS with the capability of screening in the order of 20,000 compounds per screening campaign, two to three times per seven-day week.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
High-content imaging has produced greater insights into the complexities of cell biology. The ability to characterise specific phenotypes, as demonstrated by Rosenthal and Ng, provides a powerful ...tool for elucidating mechanisms of action and resistance, illustrating that high-content imaging in malaria research is only limited by our creativity.
•Leishmaniasis remains a neglected tropical disease, with a high unmet medical need.•Therapeutic efficacy varies depending on the species of Leishmania parasite.•Leads are discovered through ...phenotypic high-throughput screening approaches.•Anti-leishmanial drug discovery has shifted to high-content screening.
Leishmaniasis, caused by the trypanosomatid protozoan Leishmania, is endemic in 98 countries worldwide, with morbidity and mortality increasing daily. Despite available drugs, leishmaniasis faces the challenge of emerging resistance and toxicity concerns for current drug regimes. Identification of anti-leishmanial compounds representing new chemistry and novel mechanisms of action is essential to populate the drug discovery pipeline. The in vitro assays currently available have shown poor translational outcomes, with high compound attrition rates. It is therefore imperative that more physiologically relevant assays are developed to identify anti-leishmanial compounds. This review provides an overview of the disease, current treatment options and compares the various technologies and assay formats currently available for leishmanial drug discovery.
This review introduces, discusses and compares preceding and recent technological advances in development and utilisation of high-throughput phenotypic screening assays to identify novel anti-leishmanial compounds.
With the increasing occurrence of drug resistance in the malaria parasite, Plasmodium falciparum, there is a great need for new and novel anti-malarial drugs. We have developed a 384-well, ...high-throughput imaging assay for the detection of new anti-malarial compounds, which was initially validated by screening a marine natural product library, and subsequently used to screen more than 3 million data points from a variety of compound sources. Founded on another fluorescence-based P. falciparum growth inhibition assay, the DNA-intercalating dye 4',6-diamidino-2-phenylindole, was used to monitor changes in parasite number. Fluorescent images were acquired on the PerkinElmer Opera High Throughput confocal imaging system and analyzed with a spot detection algorithm using the Acapella data processing software. Further optimization of this assay sought to increase throughput, assay stability, and compatibility with our high-throughput screening equipment platforms. The assay typically yielded Z'-factor values of 0.5-0.6, with signal-to-noise ratios of 12.
Human cancer cell lines are an integral part of drug discovery practices. However, modeling the complexity of cancer utilizing these cell lines on standard plastic substrata, does not accurately ...represent the tumor microenvironment. Research into developing advanced tumor cell culture models in a three-dimensional (3D) architecture that more prescisely characterizes the disease state have been undertaken by a number of laboratories around the world. These 3D cell culture models are particularly beneficial for investigating mechanistic processes and drug resistance in tumor cells. In addition, a range of molecular mechanisms deconstructed by studying cancer cells in 3D models suggest that tumor cells cultured in two-dimensional monolayer conditions do not respond to cancer therapeutics/compounds in a similar manner. Recent studies have demonstrated the potential of utilizing 3D cell culture models in drug discovery programs; however, it is evident that further research is required for the development of more complex models that incorporate the majority of the cellular and physical properties of a tumor.
Open-access drug discovery provides a substantial resource for diseases primarily affecting the poor and disadvantaged. The open-access Pathogen Box collection is comprised of compounds with ...demonstrated biological activity against specific pathogenic organisms. The supply of this resource by the Medicines for Malaria Venture has the potential to provide new chemical starting points for a number of tropical and neglected diseases, through repurposing of these compounds for use in drug discovery campaigns for these additional pathogens. We tested the Pathogen Box against kinetoplastid parasites and malaria life cycle stages
Consequently, chemical starting points for malaria, human African trypanosomiasis, Chagas disease, and leishmaniasis drug discovery efforts have been identified. Inclusive of this
biological evaluation, outcomes from extensive literature reviews and database searches are provided. This information encompasses commercial availability, literature reference citations, other aliases and ChEMBL number with associated biological activity, where available. The release of this new data for the Pathogen Box collection into the public domain will aid the open-source model of drug discovery. Importantly, this will provide novel chemical starting points for drug discovery and target identification in tropical disease research.
•Changes in the microenvironment (TME) promote drug resistance.•Cellular composition, biochemical and -biophysical parameters create tissue-specific TME.•Targeting cancer cell – TME interactions as a ...strategy to overcome drug resistance.•Modelling the TME and TME changes is essential for early drug discovery assays.•Challenges of 3D in vitro tumour models with increased complexity.
The tumour microenvironment (TME) comprises not only malignant and non-malignant cells, but also the extracellular matrix (ECM), secreted factors, and regulators of cellular functions. In addition to genetic alterations, changes of the biochemical/biophysical properties or cellular composition of the TME have been implicated in drug resistance. Here, we review the composition of the ECM and different elements of the TME contributing to drug resistance, including soluble factors, hypoxia, extracellular acidity, and cell adhesion properties. We discuss selected approaches for modelling the TME, current progress, and their use in low-and high-throughput assays for preclinical studies. Lastly, we summarise the status quo of advanced 3D cancer models compatible with high-throughput screening (HTS), the technical practicalities and challenges.
The tumour microenvironment, and changes thereof, are key aspects in mediating drug resistance. Capturing these factors in representative, disease-specific in vitro models in early drug discovery efforts would improve drug discovery outcomes; however, to date, this remains challenging.
The discovery of new antimalarial drugs able to target both the asexual and gametocyte stages ofPlasmodium falciparumis critical to the success of the malaria eradication campaign. We have developed ...and validated a robust, rapid, and cost-effective high-throughput reporter gene assay to identify compounds active against late-stage (stage IV and V) gametocytes. The assay, which is suitable for testing compound activity at incubation times up to 72 h, demonstrates excellent quality and reproducibility, with averageZ' values of 0.85 ± 0.01. We used the assay to screen more than 10,000 compounds from three chemically diverse libraries. The screening outcomes highlighted the opportunity to use collections of compounds with known activity against the asexual stages of the parasites as a starting point for gametocytocidal activity detection in order to maximize the chances of identifying gametocytocidal compounds. This assay extends the capabilities of our previously reported luciferase assay, which tested compounds against early-stage gametocytes, and opens possibilities to profile the activities of gametocytocidal compounds over the entire course of gametocytogenesis.
The advent of Plasmodium falciparum (Pf) in vitro culturing opened the door for malaria research, yielding dramatic advancements in our understanding of the parasite. However, fundamental foundations ...taken for granted in our research endeavors can unknowingly be an Achilles heel, resulting in potential misdirection. In relation to malaria research, this could be our nonquestioning acceptance of routine in vitro culture of Pf. There is nothing routine or straightforward regarding the dynamic and intimate relationship between the parasite and the in vitro environment. Here, we discuss recent studies demonstrating the impact that slight variations in in vitro Pf culture parameters can have on scientific conclusions. We reason that culture conditions should be re-established as a primary consideration in in vitro malaria experimentation.
In vitro cultivation of Pf is essential for basic biological and drug-discovery malaria research.
Studies on the effects of in vitro culturing techniques and reagents have highlighted effects on parasite biology at the molecular level. These effects may not always be obvious within a study but do have consequences in relation to data obtained and therefore the analysis and interpretation.
Detailed reporting of culturing protocols is essential, and comparisons between alternative protocols within multicentre, multiresearch studies should be acknowledged and discussed.
The influence of in vitro culturing conditions of Pf within the study of artemisinin resistance is of particular interest as it appears to influence genetic markers obtained for artemisinin-resistant parasites generated by drug selection in vitro.
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