Synthesis and Systematic Investigation of Lepidiline A and Its Gold(I), Silver(I), and Copper(I) Complexes Using In Vitro Cancer Models and Multipotent Stem Cells.

The imidazole alkaloid lepidiline A from the root of Lepidium meyenii has a moderate to low in vitro anticancer effect. Our aim was to extend cytotoxicity investigations against a panel of cancer cells, including multidrug-resistant cancer cells, and multipotent stem cells. Lepidiline A is a N-heterocyclic carbene precursor, therefore a suitable ligand source for metal complexes. Thus, we synthesized lepidiline A and its copper(I), gold(I), and silver(I) complexes and tested them against ovarian, gastrointestinal, breast, and uterine cancer cells and bone marrow-derived and adipose-derived mesenchymal stem cells. Lepidiline A and its copper complex demonstrated moderate cytotoxicity, while silver and gold complexes exhibited significantly enhanced and consistent cytotoxicity against both cancer and stem cell lines. ABCB1 in the multidrug-resistant uterine sarcoma line conferred significant resistance against lepidiline A and the copper-lepidiline A complex, but not against the silver and gold complexes. Our results indicate that only the copper complex induced a significant and universal increase in the production of reactive oxygen species within cells. In summary, binding of metal ions to lepidiline A results in enhanced cytotoxicity with the nature of the metal ion playing a critical role in determining its properties.

Reproducibility analysis of bioimpedance-based self-developed live cell assays.

Bioimpedance spectrum (BIS) measurements have a great future in in vitro experiments, meeting all the requirements for non-destructive and label-free methods. Nevertheless, a real basic research can provide the necessary milestones to achieve the success of the method. In this paper a self-developed technology-based approach for in vitro assays is proposed. Authors invented a special graphene-based measuring plate in order to assess the high sensitivity and reproducibility of introduced technique. The design of the self-produced BIS plates maximizes the detection capacity of qualitative changes in cell culture and it is robust against physical effects and artifacts. The plates do not influence the viability and proliferation, however the results are robust, stable and reproducible regardless of when and where the experiments are carried out. In this study, physiological saline concentrations, two cancer and stem cell lines were utilized. All the results were statistically tested and confirmed. The findings of the assays show, that the introduced BIS technology is appropriate to be used in vitro experiments with high efficacy. The experimental results demonstrate high correlation values across the replicates, and the model parameters suggested that the characteristic differences among the various cell lines can be detected using appropriate hypothesis tests.

Mesenchymal Stem Cells Increase Drug Tolerance of A431 Cells Only in 3D Spheroids, Not in 2D Co-Cultures.

Mesenchymal stem cells (MSCs) are an integral part of the tumor microenvironment (TME); however, their role is somewhat controversial: conflicting reports suggest that, depending on the stage of tumor development, MSCs can either support or suppress tumor growth and spread. Additionally, the influence of MSCs on drug resistance is also ambiguous. Previously, we showed that, despite MSCs proliferating significantly more slowly than cancer cells, there are chemotherapeutic drugs which proved to be similarly toxic to both cell types. Here we established 2D co-cultures and 3D co-culture spheroids from different ratios of GFP-expressing, adipose tissue-derived MSCs and A431 epidermoid carcinoma cells tagged with mCherry to investigate the effect of MSCs on cancer cell growth, survival, and drug sensitivity. We examined the cytokine secretion profile of mono- and co-cultures, explored the inner structure of the spheroids, applied MSC-(nutlin-3) and cancer cell-targeting (cisplatin) treatments separately, monitored the response with live-cell imaging and identified a new, double-fluorescent cell type emerging from these cultures. In 2D co-cultures, no effect on proliferation or drug sensitivity was observed, regardless of the changes in cytokine secretion induced by the co-culture. Conversely, 3D spheroids developed a unique internal structure consisting of MSCs, which significantly improved cancer cell survival and resilience to treatment, suggesting that physical proximity and cell-cell connections are required for MSCs to considerably affect nearby cancer cells. Our results shed light on MSC-cancer cell interactions and could help design new, better treatment options for tumors.

Comparison of Different Clinical Chemotherapeutical Agents' Toxicity and Cell Response on Mesenchymal Stem Cells and Cancer Cells.

Mesenchymal stem cells (MSCs) or fibroblasts are one of the most abundant cell types in the tumor microenvironment (TME) exerting various anti- and pro-apoptotic effects during tumorigenesis, invasion, and drug treatment. Despite the recently discovered importance of MSCs in tumor progression and therapy, the response of these cells to chemotherapeutics compared to cancer cells is rarely investigated. A widely accepted view is that these naive MSCs have higher drug tolerance than cancer cells due to a significantly lower proliferation rate. Here, we examine the differences and similarities in the sensitivity of MSCs and cancer cells to nine diverse chemotherapy agents and show that, although MSCs have a slower cell cycle, these cells are still sensitive to various drugs. Surprisingly, MSCs showed similar sensitivity to a panel of compounds, however, suffered fewer DNA double-stranded breaks, did not enter into a senescent state, and was virtually incapable of apoptosis. Our results suggest that MSCs and cancer cells have different cell fates after drug treatment, and this could influence therapy outcome. These findings could help design drug combinations targeting both MSCs and cancer cells in the TME.

[Development of novel treatment strategies for drug resistant cancer]. / Új stratégiák fejlesztése a gyógyszerrezisztens daganatok kezeléséhez.

There are about 14 million new cancer cases and 8 million deaths every year. Every second man and one in every three women will get cancer during their lifetimes. Following decades of steady increase, death rates have stabilized due to increased awareness and prevention, early detection, and the emergence of more effective therapy. Yet despite all the advances cancer remains a major killer. Despite improved therapies, nearly all current treatments face the same problem: for many patients, they ultimately stop working. Therapy resistance is the ultimate challenge facing cancer researchers and patients today. In this review we present an overview of the most important resistance mechanisms, discussing progress in therapies designed to prevent or overcome anticancer therapy resistance. Finally, we present recent findings from our own laboratory on the development of new experimental models and new therapeutic approaches to combat multidrug resistant cancer.

YAP, ΔNp63, and ß-Catenin Signaling Pathways Are Involved in the Modulation of Corneal Epithelial Stem Cell Phenotype Induced by Substrate Stiffness.

Recent studies have established that the phenotype of epithelial stem cells residing in the corneal periphery (the limbus) depends on this niche's distinct biomechanical properties. However, the signaling pathways underlying this dependency are still poorly understood. To address this issue, we investigated the effect of substrate stiffness on the migration, proliferation, and molecular phenotype of human limbal epithelial stem cells (LESCs). Specifically, we demonstrated that cells grown on collagen-based substrates with limbus-like compliance showed higher proliferation and stratification and lower migration capabilities, as well as higher levels of pro-proliferative markers Ki67 and ß-Catenin, and LESC markers ΔNp63, ABCG2, and CK15. In contrast, cells on stiffer substrates lost these stem/progenitor cell markers, but instead expressed the key mechanotransduction factor YAP, as well as elevated levels of BMP4, a promotor of cell differentiation known to be negatively regulated by Wnt/ß-Catenin signaling. This data allowed us to propose a new model that integrates the various molecular pathways involved in LESC response to substrate stiffness. This model will potentially be a useful guide to future research on the mechanisms underlying LESC loss following fibrosis-causing injuries.

Oligodendrocyte- and Neuron-Specific Nogo-A Restrict Dendritic Branching and Spine Density in the Adult Mouse Motor Cortex.

Nogo-A has been well described as a myelin-associated inhibitor of neurite outgrowth and functional neuroregeneration after central nervous system (CNS) injury. Recently, a new role of Nogo-A has been identified as a negative regulator of synaptic plasticity in the uninjured adult CNS. Nogo-A is present in neurons and oligodendrocytes. However, it is yet unclear which of these two pools regulate synaptic plasticity. To address this question we used newly generated mouse lines in which Nogo-A is specifically knocked out in (1) oligodendrocytes (oligoNogo-A KO) or (2) neurons (neuroNogo-A KO). We show that both oligodendrocyte- and neuron-specific Nogo-A KO mice have enhanced dendritic branching and spine densities in layer 2/3 cortical pyramidal neurons. These effects are compartmentalized: neuronal Nogo-A affects proximal dendrites whereas oligodendrocytic Nogo-A affects distal regions. Finally, we used two-photon laser scanning microscopy to measure the spine turnover rate of adult mouse motor cortex layer 5 cells and find that both Nogo-A KO mouse lines show enhanced spine remodeling after 4 days. Our results suggest relevant control functions of glial as well as neuronal Nogo-A for synaptic plasticity and open new possibilities for more selective and targeted plasticity enhancing strategies.

Nogo-A deletion increases the plasticity of the optokinetic response and changes retinal projection organization in the adult mouse visual system.

The inhibitory action of Nogo-A on axonal growth has been well described. However, much less is known about the effects that Nogo-A could exert on the plasticity of neuronal circuits under physiological conditions. We investigated the effects of Nogo-A knock-out (KO) on visual function of adult mice using the optokinetic response (OKR) and the monocular deprivation (MD)-induced OKR plasticity and analyzed the anatomical organization of the eye-specific retinal projections. The spatial frequency sensitivity was higher in intact Nogo-A KO than in wild-type (WT) mice. After MD, Nogo-A KO mice reached a significantly higher spatial frequency and contrast sensitivity. Bilateral ablation of the visual cortex did not affect the OKR sensitivity before MD but reduced the MD-induced enhancement of OKR by approximately 50% in Nogo-A KO and WT mice. These results suggest that cortical and subcortical brain structures contribute to the OKR plasticity. The tracing of retinal projections to the dorsal lateral geniculate nucleus (dLGN) revealed that the segregation of eye-specific terminals was decreased in the adult Nogo-A KO dLGN compared with WT mice. Strikingly, MD of the right eye led to additional desegregation of retinal projections in the left dLGN of Nogo-A KO but not in WT mice. In particular, MD promoted ectopic varicosity formation in Nogo-A KO dLGN axons. The present data show that Nogo-A restricts visual experience-driven plasticity of the OKR and plays a role in the segregation and maintenance of retinal projections to the brain.

Lack of Rybp in Mouse Embryonic Stem Cells Impairs Cardiac Differentiation.

Ring1 and Yy1 binding protein (Rybp) has been implicated in transcriptional regulation, apoptotic signaling and as a member of the polycomb repressive complex 1, it has an important function in regulating pluripotency and differentiation of embryonic stem cells (ESCs). Earlier, we had proved that Rybp plays an essential role in mouse embryonic and central nervous system development. This work identifies Rybp, as a critical regulator of heart development. Rybp is readily detectable in the developing mouse heart from day 8.5 of embryonic development. Prominent Rybp expression persists during all embryonic stages, and Rybp marks differentiated cell types of the heart. By utilizing rybp null ESCs in an in vitro cardiac differentiation assay, we found that rybp null ESCs do not form rhythmically beating cardiomyocytes (CMCs). Gene expression profiles revealed a downregulation of cardiac terminal and upregulation of germline-specific markers in the rybp null CMCs. Furthermore, transcriptome analysis uncovered a number of novel candidate target genes regulated by Rybp. Among these are several that are important in cardiac development and contractility such as Plagl1, Isl1, and Tnnt2. Importantly, forced expression of rybp in rybp-deficient ESCs by a lentiviral vector was able to rescue the mutant phenotype. Our data provide evidence for a previously unrecognized function of Rybp in heart development and point out the importance of germ cell lineage gene silencing during somatic differentiation.