The tyrosine kinase inhibitor Nintedanib induces lysosomal dysfunctionality: Role of protonation-dependent crystallization processes.

Nintedanib (NIN), a multi-tyrosine kinase inhibitor clinically approved for idiopathic pulmonary fibrosis and lung cancer, is characterized by protonation-dependent lysosomotropic behavior and appearance of lysosome-specific fluorescence emission properties. Here we investigate whether spontaneous formation of a so far unknown NIN matter within the acidic cell compartment is underlying these unexpected emissive properties and investigate the consequences on lysosome functionality. Lysosomes of cells treated with NIN, but not non-protonatable NIN derivatives, exhibited lysosome-associated birefringence signals co-localizing with the NIN-derived fluorescence emission. Sensitivity of both parameters towards vATPase inhibitors confirmed pH-dependent, spontaneous adoption of novel crystalline NIN structures in lysosomes. Accordingly, NIN crystallization from buffer solutions resulted in formation of multiple crystal polymorphs with pH-dependent fluorescence properties. Cell-free crystals grown at lysosomal-like pH conditions resembled NIN-treated cell lysosomes concerning fluorescence pattern, photobleaching dynamics, and Raman spectra. However, differences in birefringence intensity and FAIM-determined anisotropy, as well as predominant association with (intra)lysosomal membrane structures, suggested formation of a semi-solid NIN crystalline matter in acidic lysosomes. Despite comparable target kinase inhibition, NIN, but not its non-protonatable derivatives, impaired lysosomal functionality, mediated massive cell vacuolization, enhanced autophagy, deregulated lipid metabolism, and induced atypical phospholipidosis. Moreover, NIN exerted distinct phototoxicity, strictly dependent on lysosomal microcrystallization events. The spontaneous formation of NIN crystalline structures was also observable in the gut mucosa of orally NIN-treated mice. Summarizing, the here-described kinase inhibition-independent impact of NIN on lysosomal functionality mediates several of its cell biological activities and might contribute to NIN adverse effects.

Organotypic human skin culture models constructed with senescent fibroblasts show hallmarks of skin aging.

Skin aging is driven by intrinsic and extrinsic factors impacting on skin functionality with progressive age. One factor of this multifaceted process is cellular senescence, as it has recently been identified to contribute to a declining tissue functionality in old age. In the skin, senescent cells have been found to markedly accumulate with age, and thus might impact directly on skin characteristics. Especially the switch from young, extracellular matrix-building fibroblasts to a senescence-associated secretory phenotype (SASP) could alter the microenvironment in the skin drastically and therefore promote skin aging. In order to study the influence of senescence in human skin, 3D organotypic cultures are a well-suited model system. However, only few "aged" skin- equivalent (SE) models are available, requiring complex and long-term experimental setups. Here, we adapted a previously published full-thickness SE model by seeding increasing ratios of stress-induced premature senescent versus normal fibroblasts into the collagen matrix, terming these SE "senoskin". Immunohistochemistry stainings revealed a shift in the balance between proliferation (Ki67) and differentiation (Keratin 10 and Filaggrin) of keratinocytes within our senoskin equivalents, as well as partial impairment of skin barrier function and changed surface properties. Monitoring of cytokine levels of known SASP factors confirmedly showed an upregulation in 2D cultures of senescent cells and at the time of seeding into the skin equivalent. Surprisingly, we find a blunted response of cytokines in the senoskin equivalent over time during 3D differentiation.

Raman fingerprints as promising markers of cellular senescence and aging.

Due to our aging population, understanding of the underlying molecular mechanisms constantly gains more and more importance. Senescent cells, defined by being irreversibly growth arrested and associated with a specific gene expression and secretory pattern, accumulate with age and thus contribute to several age-related diseases. However, their specific detection, especially in vivo, is still a major challenge. Raman microspectroscopy is able to record biochemical fingerprints of cells and tissues, allowing a distinction between different cellular states, or between healthy and cancer tissue. Similarly, Raman microspectroscopy was already successfully used to distinguish senescent from non-senescent cells, as well as to investigate other molecular changes that occur at cell and tissue level during aging. This review is intended to give an overview about various applications of Raman microspectroscopy to study aging, especially in the context of detecting senescent cells.

Loss of the ribosomal RNA methyltransferase NSUN5 impairs global protein synthesis and normal growth.

Modifications of ribosomal RNA expand the nucleotide repertoire and thereby contribute to ribosome heterogeneity and translational regulation of gene expression. One particular m5C modification of 25S ribosomal RNA, which is introduced by Rcm1p, was previously shown to modulate stress responses and lifespan in yeast and other small organisms. Here, we report that NSUN5 is the functional orthologue of Rcm1p, introducing m5C3782 into human and m5C3438 into mouse 28S ribosomal RNA. Haploinsufficiency of the NSUN5 gene in fibroblasts from William Beuren syndrome patients causes partial loss of this modification. The N-terminal domain of NSUN5 is required for targeting to nucleoli, while two evolutionary highly conserved cysteines mediate catalysis. Phenotypic consequences of NSUN5 deficiency in mammalian cells include decreased proliferation and size, which can be attributed to a reduction in total protein synthesis by altered ribosomes. Strikingly, Nsun5 knockout in mice causes decreased body weight and lean mass without alterations in food intake, as well as a trend towards reduced protein synthesis in several tissues. Together, our findings emphasize the importance of single RNA modifications for ribosome function and normal cellular and organismal physiology.

The thiosemicarbazone Me2NNMe2 induces paraptosis by disrupting the ER thiol redox homeostasis based on protein disulfide isomerase inhibition.

Due to their high biological activity, thiosemicarbazones have been developed for treatment of diverse diseases, including cancer, resulting in multiple clinical trials especially of the lead compound Triapine. During the last years, a novel subclass of anticancer thiosemicarbazones has attracted substantial interest based on their enhanced cytotoxic activity. Increasing evidence suggests that the double-dimethylated Triapine derivative Me2NNMe2 differs from Triapine not only in its efficacy but also in its mode of action. Here we show that Me2NNMe2- (but not Triapine)-treated cancer cells exhibit all hallmarks of paraptotic cell death including, besides the appearance of endoplasmic reticulum (ER)-derived vesicles, also mitochondrial swelling and caspase-independent cell death via the MAPK signaling pathway. Subsequently, we uncover that the copper complex of Me2NNMe2 (a supposed intracellular metabolite) inhibits the ER-resident protein disulfide isomerase, resulting in a specific form of ER stress based on disruption of the Ca2+ and ER thiol redox homeostasis. Our findings indicate that compounds like Me2NNMe2 are of interest especially for the treatment of apoptosis-resistant cancer and provide new insights into mechanisms underlying drug-induced paraptosis.