Cancer cells have upregulated glycolysis compared with normal cells, which has led many to the assumption that oxidative phosphorylation (OXPHOS) is downregulated in all cancers. However, recent ...studies have shown that OXPHOS can be also upregulated in certain cancers, including leukemias, lymphomas, pancreatic ductal adenocarcinoma, high OXPHOS subtype melanoma, and endometrial carcinoma, and that this can occur even in the face of active glycolysis. OXPHOS inhibitors could therefore be used to target cancer subtypes in which OXPHOS is upregulated and to alleviate therapeutically adverse tumor hypoxia. Several drugs including metformin, atovaquone, and arsenic trioxide are used clinically for non-oncologic indications, but emerging data demonstrate their potential use as OXPHOS inhibitors. We highlight novel applications of OXPHOS inhibitors with a suitable therapeutic index to target cancer cell metabolism.
.
Although used in academic research for several decades, 3D culture models have long been regarded expensive, cumbersome and unnecessary in drug development processes. Technical advances, coupled with ...recent observations showing that gene expression in 3D is much closer to clinical expression profiles than those seen in 2D, have renewed attention and generated hope in the feasibility of maturing organotypic 3D systems to therapy test platforms with greater power to predict clinical efficacies. Here we describe a standardized setup for reproducible, easy-handling culture, treatment and routine analysis of multicellular spheroids, the classical 3D culture system resembling many aspects of the pathophysiological situation in human tumor tissue. We discuss essential conceptual and practical considerations for an adequate establishment and use of spheroid-based drug screening platforms and also provide a list of human carcinoma cell lines, partly on the basis of the NCI-DTP 60-cell line screen, that produce treatable spheroids under identical culture conditions. In contrast to many other settings with which to achieve similar results, the protocol is particularly useful to be integrated into standardized large-scale drug test routines as it requires a minimum number of defined spheroids and a limited amount of drug. The estimated time to run the complete screening protocol described herein--including spheroid initiation, drug treatment and determination of the analytical end points (spheroid integrity, and cell survival through the acid phosphatase assay)--is about 170 h. Monitoring of spheroid growth kinetics to determine growth delay and regrowth, respectively, after drug treatment requires long-term culturing (> or =14 d).
Background Hearing-impaired listeners often have difficulty understanding complex sentences. It is not clear if perceptual or cognitive deficits have more impact on reduced language processing ...abilities, and how a hearing aid might compensate for that. Methods In a prospective study with 5 hearing aid users and 5 normal hearing, age-matched participants, processing of complex sentences was investigated. Audiometric and working memory tests were performed. Subject- and object-initial sentences from the Oldenburg Corpus of Linguistically and audiologically controlled Sentences (OLACS) were presented to the participants during recording of an electroencephalogram (EEG). Results The perceptual difference between object and subject leading sentences does not lead to processing changes whereas the ambiguity in object leading sentences with feminine or neuter articles evokes a P600 potential. For hearing aid users, this P600 has a longer latency compared to normal hearing subjects. Conclusion The EEG is a suitable method for investigating differences in complex speech processing for hearing aid users. Longer P600 latencies indicate higher cognitive effort for processing complex sentences in hearing aid users.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The present article highlights the rationale, potential and flexibility of tumor spheroid mono- and cocultures for implementation into state of the art anti-cancer therapy test platforms. Unlike ...classical monolayer-based models, spheroids strikingly mirror the 3D cellular context and therapeutically relevant pathophysiological gradients of in vivo tumors. Some concepts for standardization and automation of spheroid culturing, monitoring and analysis are discussed, and the challenges to define the most convenient analytical endpoints for therapy testing are outlined. The potential of spheroids to contribute to either the elimination of poor drug candidates at the pre-animal and pre-clinical state or the identification of promising drugs that would fail in classical 2D cell assays is emphasised. Microtechnologies, in the form of micropatterning and microfluidics, are also discussed and offer the exciting prospect of standardized spheroid mass production to tackle high-throughput screening applications within the context of traditional laboratory settings. The extension towards more sophisticated spheroid coculture models which more closely reflect heterologous tumor tissues composed of tumor and various stromal cell types is also covered. Examples are given with particular emphasis on tumor-immune cell cocultures and their usefulness for testing novel immunotherapeutic treatment strategies. Finally, tumor cell heterogeneity and the extraordinary possibilities of putative cancer stem/tumor-initiating cell populations that can be maintained and expanded in sphere-forming assays are introduced. The relevance of the cancer stem cell hypothesis for cancer cure is highlighted, with the respective sphere cultures being envisioned as an integral tool for next generation drug development offensives.
Dictyostelium discoideum Sey1 is the single ortholog of mammalian atlastin 1–3 (ATL1‐3), which are large homodimeric GTPases mediating homotypic fusion of endoplasmic reticulum (ER) tubules. In this ...study, we generated a D. discoideum mutant strain lacking the sey1 gene and found that amoebae deleted for sey1 are enlarged, but grow and develop similarly to the parental strain. The ∆sey1 mutant amoebae showed an altered ER architecture, and the tubular ER network was partially disrupted without any major consequences for other organelles or the architecture of the secretory and endocytic pathways. Macropinocytic and phagocytic functions were preserved; however, the mutant amoebae exhibited cumulative defects in lysosomal enzymes exocytosis, intracellular proteolysis, and cell motility, resulting in impaired growth on bacterial lawns. Moreover, ∆sey1 mutant cells showed a constitutive activation of the unfolded protein response pathway (UPR), but they still readily adapted to moderate levels of ER stress, while unable to cope with prolonged stress. In D. discoideum ∆sey1 the formation of the ER‐associated compartment harbouring the bacterial pathogen Legionella pneumophila was also impaired. In the mutant amoebae, the ER was less efficiently recruited to the “Legionella‐containing vacuole” (LCV), the expansion of the pathogen vacuole was inhibited at early stages of infection and intracellular bacterial growth was reduced. In summary, our study establishes a role of D. discoideum Sey1 in ER architecture, proteolysis, cell motility and intracellular replication of L. pneumophila.
Dictyostelium discoideum Sey1 is the single ortholog of mammalian atlastin 1‐3, which are large homodimeric GTPases mediating fusion of tubular endoplasmic reticulum (ER). Mutant amoebae lacking Sey1 grow and develop similarly to the parental strain, but show an aberrant ER architecture, defects in proteolytic processes and diminished cell motility. In ∆sey1 amoebae, the recruitment of ER to the unique vacuole harboring the pathogen Legionella pneumophila is reduced, expansion of the ER‐associated vacuole is defective, and intracellular bacterial growth is impaired.
is the causative agent of a severe pneumonia called Legionnaires' disease. The environmental bacterium replicates in free-living amoebae as well as in lung macrophages in a distinct compartment, the
...-containing vacuole (LCV). The LCV communicates with a number of cellular vesicle trafficking pathways and is formed by a plethora of secreted bacterial effector proteins, which target host cell proteins and lipids. Phosphoinositide (PI) lipids are pivotal determinants of organelle identity, membrane dynamics and vesicle trafficking. Accordingly, eukaryotic cells tightly regulate the production, turnover, interconversion, and localization of PI lipids.
modulates the PI pattern in infected cells for its own benefit by (i) recruiting PI-decorated vesicles, (ii) producing effectors acting as PI interactors, phosphatases, kinases or phospholipases, and (iii) subverting host PI metabolizing enzymes. The PI conversion from PtdIns(3)
to PtdIns(4)
represents a decisive step during LCV maturation. In this review, we summarize recent progress on elucidating the strategies, by which
subverts host PI lipids to promote LCV formation and intracellular replication.
Environmental bacteria of the genus
naturally parasitize free-living amoebae. Upon inhalation of bacteria-laden aerosols, the opportunistic pathogens grow intracellularly in alveolar macrophages and ...can cause a life-threatening pneumonia termed Legionnaires' disease. Intracellular replication in amoebae and macrophages takes place in a unique membrane-bound compartment, the
-containing vacuole (LCV). LCV formation requires the bacterial Icm/Dot type IV secretion system, which translocates literally hundreds of "effector" proteins into host cells, where they modulate crucial cellular processes for the pathogen's benefit. The mechanism of LCV formation appears to be evolutionarily conserved, and therefore, amoebae are not only ecologically significant niches for
spp., but also useful cellular models for eukaryotic phagocytes. In particular,
and
emerged over the last years as versatile and powerful models. Using genetic, biochemical and cell biological approaches, molecular interactions between amoebae and
have recently been investigated in detail with a focus on the role of phosphoinositide lipids, small and large GTPases, autophagy components and the retromer complex, as well as on bacterial effectors targeting these host factors.
Tumour hypoxia renders cancer cells resistant to cancer therapy, resulting in markedly worse clinical outcomes. To find clinical candidate compounds that reduce hypoxia in tumours, we conduct a ...high-throughput screen for oxygen consumption rate (OCR) reduction and identify a number of drugs with this property. For this study we focus on the anti-malarial, atovaquone. Atovaquone rapidly decreases the OCR by more than 80% in a wide range of cancer cell lines at pharmacological concentrations. In addition, atovaquone eradicates hypoxia in FaDu, HCT116 and H1299 spheroids. Similarly, it reduces hypoxia in FaDu and HCT116 xenografts in nude mice, and causes a significant tumour growth delay when combined with radiation. Atovaquone is a ubiquinone analogue, and decreases the OCR by inhibiting mitochondrial complex III. We are now undertaking clinical studies to assess whether atovaquone reduces tumour hypoxia in patients, thereby increasing the efficacy of radiotherapy.
Legionella pneumophila can cause Legionnaires' disease and replicates intracellularly in a distinct Legionella-containing vacuole (LCV). LCV formation is a complex process that involves a plethora of ...type IV-secreted effector proteins. The effector RidL binds the Vps29 retromer subunit, blocks retrograde vesicle trafficking, and promotes intracellular bacterial replication. Here, we reveal that the 29-kDa N-terminal domain of RidL (RidL
) adopts a "foot-like" fold comprising a protruding β-hairpin at its "heel". The deletion of the β-hairpin, the exchange to Glu of Ile
in the β-hairpin, or Leu
in Vps29 abolishes the interaction in eukaryotic cells and in vitro. RidL
or RidL displace the Rab7 GTPase-activating protein (GAP) TBC1D5 from the retromer and LCVs, respectively, and TBC1D5 promotes the intracellular growth of L. pneumophila. Thus, the hydrophobic β-hairpin of RidL is critical for binding of the L. pneumophila effector to the Vps29 retromer subunit and displacement of the regulator TBC1D5.
PDF
