Drug development is a lengthy and costly process that proceeds through several stages from target identification to lead discovery and optimization, preclinical validation and clinical trials culminating in approval for clinical use. An important step in this process is high-throughput screening (HTS) of small compound libraries for lead identification. Currently, the majority of cell-based HTS is being carried out on cultured cells propagated in two-dimensions (2D) on plastic surfaces optimized for tissue culture. At the same time, compelling evidence suggests that cells cultured in these non-physiological conditions are not representative of cells residing in the complex microenvironment of a tissue. This discrepancy is thought to be a significant contributor to the high failure rate in drug discovery, where only a low percentage of drugs investigated ever make it through the gamut of testing and approval to the market. Thus, three-dimensional (3D) cell culture technologies that more closely resemble in vivo cell environments are now being pursued with intensity as they are expected to accommodate better precision in drug discovery. Here we will review common approaches to 3D culture, discuss the significance of 3D cultures in drug resistance and drug repositioning and address some of the challenges of applying 3D cell cultures to high-throughput drug discovery.

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Three-Dimensional in Vitro Cell Culture Models in Drug Discovery and Drug Repositioning.

Cell culture is an important and necessary process in drug discovery, cancer research, as well as stem cell study. Most cells are currently cultured using two-dimensional (2D) methods but new and improved methods that implement three-dimensional (3D) cell culturing techniques suggest compelling evidence that much more advanced experiments can be performed yielding valuable insights. When performing 3D cell culture experiments, the cell environment can be manipulated to mimic that of a cell in vivo and provide more accurate data about cell-to-cell interactions, tumor characteristics, drug discovery, metabolic profiling, stem cell research, and other types of diseases. Scaffold based techniques such as hydrogel-based support, polymeric hard material-based support, hydrophilic glass fiber, and organoids are employed, and each provide their own advantages and applications. Likewise, there are also scaffold free techniques used such as hanging drop microplates, magnetic levitation, and spheroid microplates with ultra-low attachment coating. 3D cell culture has the potential to provide alternative ways to study organ behavior via the use of organoids and is expected to eventually bridge the gap between 2D cell culture and animal models. The present review compares 2D cell culture to 3D cell culture, provides the details surrounding the different 3D culture techniques, as well as focuses on the present and future applications of 3D cell culture.

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Is It Time to Start Transitioning From 2D to 3D Cell Culture

Adr1p Carbon source-responsive zinc-finger transcription factor, required for transcription of the glucose-repressed gene ADH2, of peroxisomal protein genes, and of genes required for ethanol, glycerol, and fatty acid utilization (Direct: ChIP-on-chip) Cat8p derepression of a variety of genes under non-fermentative growth conditions, active after diauxic shift, binds carbon source responsive elements Hap4p Subunit of the heme-activated, glucose-repressed Hap2p/3p/4p/5p CCAAT-binding complex, a transcriptional activator and global regulator of respiratory gene expression; provides the principal activation function of the complex Indirect: Microarrays-Wild type vs. TF mutant) Stp1/Stp2 Homologous transcription factors, activated by proteolytic processing in response to signals from the SPS sensor system for external amino acids; activates transcription of amino acid permease genes and may have a role in tRNA processing

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전사인자(transcription factor)와 기관지천식

The hypoxic tumor microenvironment: A driving force for breast cancer progression

This review addresses the oncopharmacological properties of curcumin at the molecular level. First, the interactions between curcumin and its molecular targets are addressed on the basis of curcumin's distinct chemical properties, which include H-bond donating and accepting capacity of the β-dicarbonyl moiety and the phenylic hydroxyl groups, H-bond accepting capacity of the methoxy ethers, multivalent metal and nonmetal cation binding properties, high partition coefficient, rotamerization around multiple C-C bonds, and the ability to act as a Michael acceptor. Next, the in vitro chemical stability of curcumin is elaborated in the context of its susceptibility to photochemical and chemical modification and degradation (e.g., alkaline hydrolysis). Specific modification and degradatory pathways are provided, which mainly entail radical-based intermediates, and the in vitro catabolites are identified. The implications of curcumin's (photo)chemical instability are addressed in light of pharmaceutical curcumin preparations, the use of curcumin analogues, and implementation of nanoparticulate drug delivery systems. Furthermore, the pharmacokinetics of curcumin and its most important degradation products are detailed in light of curcumin's poor bioavailability. Particular emphasis is placed on xenobiotic phase I and II metabolism as well as excretion of curcumin in the intestines (first pass), the liver (second pass), and other organs in addition to the pharmacokinetics of curcumin metabolites and their systemic clearance. Lastly, a summary is provided of the clinical pharmacodynamics of curcumin followed by a detailed account of curcumin's direct molecular targets, whereby the phenotypical/biological changes induced in cancer cells upon completion of the curcumin-triggered signaling cascade(s) are addressed in the framework of the hallmarks of cancer. The direct molecular targets include the ErbB family of receptors, protein kinase C, enzymes involved in prostaglandin synthesis, vitamin D receptor, and DNA.

The Molecular Basis for the Pharmacokinetics and Pharmacodynamics of Curcumin and Its Metabolites in Relation to Cancer

Increasing evidence suggests that ion channels and pumps not only regulate membrane potential, ion homeostasis, and electric signaling in excitable cells but also play important roles in cell proliferation, migration, apoptosis and differentiation. Consistent with a role in cell signaling, channel proteins and ion pumps can form macromolecular complexes with growth factors, and cell adhesion and other signaling molecules. And while cancer is still not being cataloged as a channelopathy, as the non-traditional roles of ion pumps and channels are being recognized, it is increasingly being suggested that ion channels and ion pumps contribute to cancer progression. Cancer cell migration requires the regulation of adhesion complexes between migrating cells and surrounding extracellular matrix (ECM) proteins. Cell movement along solid surfaces requires a sequence of cell protrusions and retractions that mainly depend on regulation of the actin cytoskeleton along with contribution of microtubules and molecular motor proteins such as mysoin. This process is triggered and modulated by a combination of environmental signals, which are sensed and integrated by membrane receptors, including integrins and cadherins. Membrane receptors transduce these signals into downstream signaling pathways, often involving the Rho GTPase protein family. These pathways regulate the cytoskeletal rearrangements necessary for proper timing of adhesion, contraction and detachment of cells in order to find their way through extracellular spaces. Migration and adhesion involve continuous modulation of cell motility, shape and volume, in which ion channels and pumps play major roles. Research on cancer cells suggests that certain ion channels may be involved in aberrant tumor growth and channel inhibitors often lead to growth arrest. This review will describe recent research into the role of ion pumps and ion channels in cell migration and adhesion, and how they may contribute to tumor development.

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Cancer as a channelopathy: ion channels and pumps in tumor development and progression

Traditional two-dimensional (2D) cell culture systems have contributed tremendously to our understanding of cancer biology but have significant limitations in mimicking in vivo conditions such as the tumor microenvironment. In vitro, three-dimensional (3D) cell culture models represent a more accurate, intermediate platform between simplified 2D culture models and complex and expensive in vivo models. 3D in vitro models can overcome 2D in vitro limitations caused by the oversupply of nutrients, and unphysiological cell-cell and cell-material interactions, and allow for dynamic interactions between cells, stroma, and extracellular matrix. In addition, 3D cultures allow for the development of concentration gradients, including oxygen, metabolites, and growth factors, with chemical gradients playing an integral role in many cellular functions ranging from development to signaling in normal epithelia and cancer environments in vivo. Currently, the most common matrices used for 3D culture are biologically derived materials such as matrigel and collagen. However, in recent years, more defined, synthetic materials have become available as scaffolds for 3D culture with the advantage of forming well-defined, designed, tunable materials to control matrix charge, stiffness, porosity, nanostructure, degradability, and adhesion properties, in addition to other material and biological properties. One important area of synthetic materials currently available for 3D cell culture is short sequence, self-assembling peptide hydrogels. In addition to the review of recent work toward the control of material, structure, and mechanical properties, we will also discuss the biochemical functionalization of peptide hydrogels and how this functionalization, coupled with desired hydrogel material characteristics, affects tumor cell behavior in 3D culture.

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Peptide hydrogels - versatile matrices for 3D cell culture in cancer medicine

Epithelial-mesenchymal transition (EMT) and cell transformation have been well-documented in multiple cancer cell models and are believed to be one of the earliest events in tumor progression. Genetic and epigenetic modifications shift cells toward either end of the EMT spectrum, and can be influenced by the microenvironment surrounding a tumor. EMT and mesenchymal-epithelial transition (MET) are critical to normal function and development and an intricate network of transcription factors and transcriptional regulators tightly regulates these processes. As evidenced in normal and transformed cell lines, many signaling pathways trigger EMT during development and differentiation. The signaling pathways include those triggered by different members of the transforming growth factor (TGF) superfamily, epidermal growth factor (EGF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), hypoxia-inducible factor (HIF), Wnt, Notch, and many others. Functional redundancies allow cells to undergo EMT even if these key transcriptional regulators are lacking, but these same redundancies also make these pathways particularly susceptible to gain-of-function mutations or constitutive signal activation; the “forced” transition toward either a mesenchymal or epithelial phenotype.

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Crosstalk of Oncogenic Signaling Pathways during Epithelial–Mesenchymal Transition

The Na,K-ATPase classically serves as an ion pump creating an electrochemical gradient across the plasma membrane that is essential for transepithelial transport, nutrient uptake and membrane potential. In addition, Na,K-ATPase also functions as a receptor, a signal transducer and a cell adhesion molecule. With such diverse roles, it is understandable that the Na,K-ATPase subunits, the catalytic alpha-subunit, the beta-subunit and the FXYD proteins, are controlled extensively during development and to accommodate physiological needs. The spatial and temporal expression of Na,K-ATPase is partially regulated at the transcriptional level. Numerous transcription factors, hormones, growth factors, lipids and extracellular stimuli modulate the transcription of the Na,K-ATPase subunits. Moreover, epigenetic mechanisms also contribute to the regulation of Na,K-ATPase expression. With the ever growing knowledge about diseases associated with the malfunction of Na,K-ATPase, this review aims at summarizing the best-characterized transcription regulators that modulate Na,K-ATPase subunit levels. As abnormal expression of Na,K-ATPase subunits have been observed in many carcinoma, we will also discuss transcription factors that are associated with epithelial-to-mesenchymal transition, a crucial step in the progression of many tumors to malignant disease.

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Sigrid A. Langhans

Papers

Three-Dimensional in Vitro Cell Culture Models in Drug Discovery and Drug Repositioning.

Cancer as a channelopathy: ion channels and pumps in tumor development and progression

Peptide hydrogels - versatile matrices for 3D cell culture in cancer medicine

Crosstalk of Oncogenic Signaling Pathways during Epithelial–Mesenchymal Transition

Transcriptional regulators of Na,K-ATPase subunits.