Pifithrin-µ Induces Stress Granule Formation, Regulates Cell Survival, and Rewires Cellular Signaling.

(1) Background: Stress granules (SGs) are cytoplasmic protein-RNA condensates that assemble in response to various insults. SG production is driven by signaling pathways that are relevant to human disease. Compounds that modulate SG characteristics are therefore of clinical interest. Pifithrin-µ is a candidate anti-tumor agent that inhibits members of the hsp70 chaperone family. While hsp70s are required for granulostasis, the impact of pifithrin-µ on SG formation is unknown. (2) Methods: Using HeLa cells as model system, cell-based assays evaluated the effects of pifithrin-µ on cell viability. Quantitative Western blotting assessed cell signaling events and SG proteins. Confocal microscopy combined with quantitative image analyses examined multiple SG parameters. (3) Results: Pifithrin-µ induced bona fide SGs in the absence of exogenous stress. These SGs were dynamic; their properties were determined by the duration of pifithrin-µ treatment. The phosphorylation of eIF2α was mandatory to generate SGs upon pifithrin-µ exposure. Moreover, the formation of pifithrin-µ SGs was accompanied by profound changes in cell signaling. Pifithrin-µ reduced the activation of 5'-AMP-activated protein kinase, whereas the pro-survival protein kinase Akt was activated. Long-term pifithrin-µ treatment caused a marked loss of cell viability. (4) Conclusions: Our study identified stress-related changes in cellular homeostasis that are elicited by pifithrin-µ. These insights are important knowledge for the appropriate therapeutic use of pifithrin-µ and related compounds.

Silica-coated LiYF4:Yb3+, Tm3+ upconverting nanoparticles are non-toxic and activate minor stress responses in mammalian cells.

Lanthanide-doped upconverting nanoparticles (UCNPs) are ideal candidates for use in biomedicine. The interaction of nanomaterials with biological systems determines whether they are suitable for use in living cells. In-depth knowledge of the nano-bio interactions is therefore a pre-requisite for the development of biomedical applications. The current study evaluates fundamental aspects of the NP-cell interface for square bipyramidal UCNPs containing a LiYF4:Yb3+, Tm3+ core and two different silica surface coatings. Given their importance for mammalian physiology, fibroblast and renal proximal tubule epithelial cells were selected as cellular model systems. We have assessed the toxicity of the UCNPs and measured their impact on the homeostasis of living non-malignant cells. Rigorous analyses were conducted to identify possible toxic and sub-lethal effects of the UCNPs. To this end, we examined biomarkers that reveal if UCNPs induce cell killing or stress. Quantitative measurements demonstrate that short-term exposure to the UCNPs had no profound effects on cell viability, cell size or morphology. Indicators of oxidative, endoplasmic reticulum, or nucleolar stress, and the production of molecular chaperones varied with the surface modification of the UCNPs and the cell type analyzed. These differences emphasize the importance of evaluating cells of diverse origin that are relevant to the intended use of the nanomaterials. Taken together, we established that short-term, our square bipyramidal UCNPs are not toxic to non-malignant fibroblast and proximal renal epithelial cells. Compared with established inducers of cellular stress, these UCNPs have minor effects on cellular homeostasis. Our results build the foundation to explore square bipyramidal UCNPs for future in vivo applications.

Oxidative stress and signaling through EGFR and PKA pathways converge on the nuclear transport factor RanBP1.

Nuclear protein trafficking requires the soluble transport factor RanBP1. The subcellular distribution of RanBP1 is dynamic, as the protein shuttles between the nucleus and cytoplasm. To date, the signaling pathways regulating RanBP1 subcellular localization are poorly understood. During interphase, RanBP1 resides mostly in the cytoplasm. We show here that oxidative stress concentrates RanBP1 in the nucleus, and our study defines the underlying mechanisms. Specifically, RanBP1's cysteine residues are not essential for its oxidant-induced relocation. Furthermore, our pharmacological approaches uncover that signaling mediated by epidermal growth factor receptor (EGFR) and protein kinase A (PKA) control RanBP1 localization during stress. In particular, pharmacological inhibitors of EGFR or PKA diminish the oxidant-dependent relocation of RanBP1. Mutant analysis identified serine 60 and tyrosine 103 as regulators of RanBP1 nuclear accumulation during oxidant exposure. Taken together, our results define RanBP1 as a target of oxidative stress and a downstream effector of EGFR and PKA signaling routes. This positions RanBP1 at the intersection of important cellular signaling circuits.

Yeast profilin mutants inhibit classical nuclear import and alter the balance between actin and tubulin levels.

Profilin is a small protein that controls actin polymerization in yeast and higher eukaryotes. In addition, profilin has emerged as a multifunctional protein that contributes to other processes in multicellular organisms. This study focuses on profilin (Pfy1) in the budding yeast Saccharomyces cerevisiae. The primary sequences of yeast Pfy1 and its metazoan orthologs diverge vastly. However, structural elements of profilin are conserved among different species. To date, the full spectrum of Pfy1 functions has yet to be defined. The current work explores the possible involvement of yeast profilin in nuclear protein import. To this end, a panel of well-characterized yeast profilin mutants was evaluated. The experiments demonstrate that yeast profilin (i) regulates nuclear protein import, (ii) determines the subcellular localization of essential nuclear transport factors, and (iii) controls the relative abundance of actin and tubulin. Together, these results define yeast profilin as a moonlighting protein that engages in multiple essential cellular activities.

Valosin containing protein (VCP): initiator, modifier, and potential drug target for neurodegenerative diseases.

The AAA+ ATPase valosin containing protein (VCP) is essential for cell and organ homeostasis, especially in cells of the nervous system. As part of a large network, VCP collaborates with many cofactors to ensure proteostasis under normal, stress, and disease conditions. A large number of mutations have revealed the importance of VCP for human health. In particular, VCP facilitates the dismantling of protein aggregates and the removal of dysfunctional organelles. These are critical events to prevent malfunction of the brain and other parts of the nervous system. In line with this idea, VCP mutants are linked to the onset and progression of neurodegeneration and other diseases. The intricate molecular mechanisms that connect VCP mutations to distinct brain pathologies continue to be uncovered. Emerging evidence supports the model that VCP controls cellular functions on multiple levels and in a cell type specific fashion. Accordingly, VCP mutants derail cellular homeostasis through several mechanisms that can instigate disease. Our review focuses on the association between VCP malfunction and neurodegeneration. We discuss the latest insights in the field, emphasize open questions, and speculate on the potential of VCP as a drug target for some of the most devastating forms of neurodegeneration.

Combining Pr3+-Doped Nanoradiosensitizers and Endogenous Protoporphyrin IX for X-ray-Mediated Photodynamic Therapy of Glioblastoma Cells.

Glioblastoma multiforme is an aggressive type of brain cancer with high recurrence rates due to the presence of radioresistant cells remaining after tumor resection. Here, we report the development of an X-ray-mediated photodynamic therapy (X-PDT) system using NaLuF4:25% Pr3+ radioluminescent nanoparticles in conjunction with protoporphyrin IX (PPIX), an endogenous photosensitizer that accumulates selectively in cancer cells. Conveniently, 5-aminolevulinic acid (5-ALA), the prodrug that is administered for PDT, is the only drug approved for fluorescence-guided resection of glioblastoma, enabling dual detection and treatment of malignant cells. NaLuF4:Pr3+ nanoparticles were synthesized and spectroscopically evaluated at a range of Pr3+ concentrations. This generated radioluminescent nanoparticles with strong emissions from the 1S0 excited state of Pr3+, which overlaps with the Soret band of PPIX to perform photodynamic therapy. The spectral overlap between the nanoparticles and PPIX improved treatment outcomes for U251 cells, which were used as a model for the thin tumor margin. In addition to sensitizing PPIX to induce X-PDT, our nanoparticles exhibit strong radiosensitizing properties through a radiation dose-enhancement effect. We evaluate the effects of the nanoparticles alone and in combination with PPIX on viability, death, stress, senescence, and proliferation. Collectively, our results demonstrate this as a strong proof of concept for nanomedicine.

Multiple pathways promote microtubule stabilization in senescent intestinal epithelial cells.

Intestinal epithelial cells are critical for gastrointestinal homeostasis. However, their function declines during aging. The aging-related loss of organ performance is largely driven by the increase in senescent cells. To date, the hallmarks and molecular mechanisms related to cellular senescence are not fully understood. Microtubules control epithelial functions, and we identified microtubule stabilization as a phenotypic marker of senescent intestinal epithelial cells. The senescence inducer determined the pathway to microtubule stabilization. Specifically, enhanced microtubule stability was associated with α-tubulin hyperacetylation or increased abundance of the microtubule-binding protein tau. We show further that overexpression of MAPT, which encodes tau, augmented microtubule stability in intestinal epithelial cells. Notably, pharmacological microtubule stabilization was sufficient to induce cellular senescence. Taken together, this study provides new insights into the molecular mechanisms that control epithelial cell homeostasis. Our results support the concept that microtubule stability serves as a critical cue to trigger intestinal epithelial cell senescence.

CD26 is a senescence marker associated with reduced immunopotency of human adipose tissue-derived multipotent mesenchymal stromal cells.

INTRODUCTION: Human mesenchymal stromal cells (MSCs) have immunomodulatory, anti-inflammatory, and tolerogenic effects. Long-term in vitro expansion of MSCs to generate clinical grade products results in the accumulation of senescent-functionally impaired MSCs. Markers to assess the 'senescent load' of MSC products are needed. METHODS: Early and late passage human adipose tissue (AT) MSCs from pediatric and adult donors were characterized using established senescent markers [i.e., MSC size, granularity, and autofluorescence by flow cytometry; ß-galactosidase staining (SA-ß-gal); CDKN2A and CDKN1A by qRT-PCR]. In gene set enrichment analysis, DPP4 (also known as adenosine deaminase complexing protein 2 or CD26) was found as a prominent dysregulated transcript that was increased in late passage MSC(AT). This was confirmed in a larger number of MSC samples by PCR, flow cytometry, Western blotting, and immunofluorescence. In vitro immunopotency assays compared the function of CD26high and CD26low MSC(AT). The effect of senolytics on the CD26high subpopulation was evaluated in senescent MSC(AT). RESULTS: Late passage MSC(AT) had a senescence transcriptome signature. DPP4 was the most differentially enriched gene in senescent MSCs. Late passage senescent MSC(AT) had higher CD26 surface levels and total protein abundance. Moreover, CD26 surface levels were higher in early passage MSC(AT) from adults compared to pediatric donors. CD26 abundance correlated with established senescence markers. CD26high MSC(AT) had reduced immunopotency compared to CD26low MSC(AT). Senolytic treatment induced MSC apoptosis, which decreased the frequencies of CD26high MSC(AT). CONCLUSIONS: DPP4 gene expression and DPP4/CD26 protein abundance are markers of replicative senescence in MSC(AT). Samples enriched in CD26high MSC(AT) have reduced immunopotency and CD26high MSCs are reduced with senolytics.

Targeting nanoparticles to malignant tumors.

Nanomaterials are at the forefront of health research and development. Among different nanomaterials, nanoparticles are especially promising for cancer theranostics. However, despite great potential, the clinical translation of nano-based applications continues to face obstacles. A major hurdle to the localized eradication of tumors is the efficient targeting of nanomaterials to the desired tissues and cells. In particular, nanoparticle properties and the route of administration impact the efficacy of precision nanomedicine. This review focuses on nanoparticles that have been produced for the detection and treatment of cancer. Common and tissue-specific barriers that limit the accumulation of nanoparticles in malignant tumors are discussed. The in-depth discussion focuses on the physicochemical properties of nanoparticles and the surface modifications that achieve efficient accumulation at tumor sites. Furthermore, limitations of current strategies and open questions are presented. The review concludes with an outlook on future directions and the trajectories that will drive the field forward to advance nano-oncology in the clinic.

Curcumin nanoformulations to combat aging-related diseases.

Aging increases the susceptibility to a diverse set of diseases and disorders, including neurodegeneration, cancer, diabetes, and arthritis. Natural compounds are currently being explored as alternative or complementary agents to treat or prevent aging-related malfunctions. Curcumin, a phytochemical isolated from the spice turmeric, has garnered great interest in recent years. With anti-oxidant, anti-inflammatory, anti-microbial, and other physiological activities, curcumin has great potential for health applications. However, the benefits of curcumin are restricted by its low bioavailability and stability in biological systems. Curcumin nanoformulations, or nano-curcumin, may overcome these limitations. This review discusses different forms of nano-curcumin that have been evaluated in vitro and in vivo to treat or prevent aging-associated health impairments. We describe current barriers for the routine use of curcumin nanoformulations in the clinic. Our review highlights outstanding questions and future work that is needed to ensure nano-curcumin is efficient and safe to lessen the burden of aging-related health problems.

The Co-Chaperone HspBP1 Is a Novel Component of Stress Granules that Regulates Their Formation.

The co-chaperone HspBP1 interacts with members of the hsp70 family, but also provides chaperone-independent functions. We report here novel biological properties of HspBP1 that are relevant to the formation of cytoplasmic stress granules (SGs). SG assembly is a conserved reaction to environmental or pathological insults and part of the cellular stress response. Our study reveals that HspBP1 (1) is an integral SG constituent, and (2) a regulator of SG assembly. Oxidative stress relocates HspBP1 to SGs, where it co-localizes with granule marker proteins and polyA-RNA. Mass spectrometry and co-immunoprecipitation identified novel HspBP1-binding partners that are critical for SG biology. Specifically, HspBP1 associates with the SG proteins G3BP1, HuR and TIA-1/TIAR. HspBP1 also interacts with polyA-RNA in vivo and binds directly RNA homopolymers in vitro. Multiple lines of evidence and single-granule analyses demonstrate that HspBP1 is crucial for SG biogenesis. Thus, HspBP1 knockdown interferes with stress-induced SG assembly. By contrast, HspBP1 overexpression promotes SG formation in the absence of stress. Notably, the hsp70-binding domains of HspBP1 regulate SG production in unstressed cells. Taken together, we identified novel HspBP1 activities that control SG formation. These features expand HspBP1's role in the cellular stress response and provide new mechanistic insights into SG biogenesis.

Nucleolar Organization and Functions in Health and Disease.

The nucleolus is a prominent, membraneless compartment found within the nucleus of eukaryotic cells. It forms around ribosomal RNA (rRNA) genes, where it coordinates the transcription, processing, and packaging of rRNA to produce ribosomal subunits. Recent efforts to characterize the biophysical properties of the nucleolus have transformed our understanding of the assembly and organization of this dynamic compartment. Indeed, soluble macromolecules condense from the nucleoplasm to form nucleoli through a process called liquid-liquid phase separation. Individual nucleolar components rapidly exchange with the nucleoplasm and separate within the nucleolus itself to form distinct subcompartments. In addition to its essential role in ribosome biogenesis, the nucleolus regulates many aspects of cell physiology, including genome organization, stress responses, senescence and lifespan. Consequently, the nucleolus is implicated in several human diseases, such as Hutchinson-Gilford progeria syndrome, Diamond-Blackfan anemia, and various forms of cancer. This Special Issue highlights new insights into the physical and molecular mechanisms that control the architecture and diverse functions of the nucleolus, and how they break down in disease.

Gold nanoclusters elicit homeostatic perturbations in glioblastoma cells and adaptive changes of lysosomes.

Unique physicochemical features place gold nanoclusters at the forefront of nanotechnology for biological and biomedical applications. To date, information on the interactions of gold nanoclusters with biological macromolecules is limited and restricts their use in living cells. Methods: Our multidisciplinary study begins to fill the current knowledge gap by focusing on lysosomes and associated biological pathways in U251N human glioblastoma cells. We concentrated on lysosomes, because they are the intracellular destination for many nanoparticles, regulate cellular homeostasis and control cell survival. Results: Quantitative data presented here show that gold nanoclusters (with 15 and 25 gold atoms), surface-modified with glutathione or PEG, did not diminish cell viability at concentrations ≤1 µM. However, even at sublethal concentrations, gold nanoclusters modulated the abundance, positioning, pH and enzymatic activities of lysosomes. Gold nanoclusters also affected other aspects of cellular homeostasis. Specifically, they stimulated the transient nuclear accumulation of TFEB and Nrf2, transcription factors that promote lysosome biogenesis and stress responses. Moreover, gold nanoclusters also altered the formation of protein aggregates in the cytoplasm. The cellular responses elicited by gold nanoclusters were largely reversible within a 24-hour period. Conclusions: Taken together, this study explores the subcellular and molecular effects induced by gold nanoclusters and shows their effectiveness to regulate lysosome biology. Our results indicate that gold nanoclusters cause homeostatic perturbations without marked cell loss. Notably, cells adapt to the challenge inflicted by gold nanoclusters. These new insights provide a framework for the further development of gold nanocluster-based applications in biological sciences.

Evaluation of Lanthanide-Doped Upconverting Nanoparticles for in Vitro and in Vivo Applications.

Because of their unique physicochemical properties, lanthanide-doped upconverting nanoparticles (Ln-UCNPs) have exceptional potential for biological applications. However, the use in biological systems is hampered by the limited understanding of their bionano interactions. Our multidisciplinary study has generated these insights through in-depth and quantitative analyses. The Ln-UCNPs examined here are spherical, monodisperse, and stable in aqueous environments. We show that Ln-UCNPs were associated with HeLa (cervical cancer) and LLC-PK1 (renal proximal tubule) cells and were nontoxic over a wide concentration range. Multiple biomarkers were assessed to monitor the cellular homeostasis in Ln-UCNP-treated cells. To this end, we evaluated the nuclear lamina, nucleoli, and nuclear transport factors. Single-cell analyses quantified the impact on Nrf2 and NF-κB, two transcription factors that control stress and immune responses. Moreover, we measured Ln-UCNP-induced changes in the abundance of molecular chaperones. Collectively, in vitro studies confirmed that Ln-UCNPs are nontoxic and trigger minor cellular stress responses. This lack of toxicity was verified in vivo, using the model organism Caenorhabditis elegans. The compatibility with biological systems prompted us to assess Ln-UCNPs as potential contrast agents for magnetic resonance imaging. We demonstrated that the Ln-UCNPs examined here were especially suitable as T2 contrast agents; they clearly outperformed the clinically used Gadovist. Taken together, our interdisciplinary work provides robust evidence for the nontoxicity of Ln-UCNPs. This sets the stage for the translation of Ln-UCNP for use in complex biological systems.

Exploring near-infrared absorbing nanocarriers to overcome cancer drug resistance.

One of the major obstacles of successful cancer therapy is cancer drug resistance. The unique tools and applications developed by nanomedicine provide new approaches to surmount this common limitation of current treatment regimens. Nanocarriers that absorb light in the near-infrared spectrum are particularly suitable for this purpose. These nanocarriers can produce heat, release drugs or stimulate the production of physiologically relevant compounds when illuminated with near-infrared light. The current review summarizes the causes contributing to cancer multidrug resistance. The major types of nanocarriers that have been developed in recent years to overcome these hurdles are described. We focus on nanoparticles that are responsive to near-infrared light and suitable to surmount cancer multidrug resistance. Our review concludes with the bottlenecks that currently restrict the use of nanocarriers in the clinic and an outlook on future directions.

The Cytoskeleton as Regulator of Cell Signaling Pathways.

During interphase, filamentous actin, microtubules, and intermediate filaments regulate cell shape, motility, transport, and interactions with the environment. These activities rely on signaling events that control cytoskeleton properties. Recent studies uncovered mechanisms that go far beyond this one-directional flow of information. Thus, the three branches of the cytoskeleton impinge on signaling pathways to determine their activities. We propose that this regulatory role of the cytoskeleton provides sophisticated mechanisms to control the spatiotemporal output and the intensity of signaling events. Specific examples emphasize these emerging contributions of the cytoskeleton to cell physiology. In our opinion, further exploration of these pathways will uncover new concepts of cellular communication that originate from the cytoskeleton.

Gold nanourchins induce cellular stress, impair proteostasis and damage RNA.

Gold nanoparticles have excellent potential for theranostic applications, but their impact on living cells is only partially understood. Many gold nanoparticles enter cells through endosomes/lysosomes which are linked to different cell organelles and compartments. Our study focuses on the unfolded protein response (UPR) in the endoplasmic reticulum (ER), cytoplasmic RNA-granules and proteostasis, because they are established indicators of cell stress and key regulators of cellular homeostasis. Using HeLa and renal proximal tubule cells as model systems, we show that gold nanourchins reduce cell proliferation, cause ER stress and impair proteostasis. Specifically, gold nanourchins activate the PERK-branch of the UPR, promote RNA oxidation, enhance P-body formation, and accumulate the oxidative stress marker Nrf2 and NFκB in nuclei. Taken together, our study demonstrates that gold nanourchins compromise ER, redox, protein, and RNA homeostasis. These insights provide new information on the cellular responses and molecular changes that gold nanourchins elicit in mammalian cells.

Cellular senescence is associated with reorganization of the microtubule cytoskeleton.

Senescent cells undergo structural and functional changes that affect essentially every aspect of cell physiology. To date, the impact of senescence on the cytoskeleton is poorly understood. This study evaluated the cytoskeleton in two independent cellular models of kidney epithelium senescence. Our work identified multiple senescence-related alterations that impact microtubules and filamentous actin during interphase. Both filamentous systems reorganized profoundly when cells became senescent. As such, microtubule stability increased during senescence, making these filaments more resistant to disassembly in the cold or by nocodazole. Microtubule stabilization was accompanied by enhanced α-tubulin acetylation on lysine 40 and the depletion of HDAC6, the major deacetylase for α-tubulin lysine 40. Rho-associated kinase Rock1 is an upstream regulator that modulates key properties of the cytoplasmic cytoskeleton. Our research shows that Rock1 concentrations were reduced significantly in senescent cells, and we revealed a mechanistic link between microtubule stabilization and Rock1 depletion. Thus, Rock1 overexpression partially restored the cold sensitivity of microtubules in cells undergoing senescence. Additional components relevant to microtubules were affected by senescence. Specifically, we uncovered the senescence-related loss of the microtubule nucleating protein γ-tubulin and aberrant formation of γ-tubulin foci. Concomitant with the alterations of microtubule and actin filaments, senescent cells displayed functional changes. In particular, cell migration was impaired significantly in senescent cells. Taken together, our study identified new senescence-associated deficiencies of the microtubule and actin cytoskeleton, provided insights into the underlying molecular mechanisms and demonstrated functional consequences that are important to the physiology and function of renal epithelial cells.