Expression Changes and Impact of the Extracellular Matrix on Etoposide Resistant Human Retinoblastoma Cell Lines.

Retinoblastoma (RB) represents the most common malignant childhood eye tumor worldwide. Several studies indicate that the extracellular matrix (ECM) plays a crucial role in tumor growth and metastasis. Moreover, recent studies indicate that the ECM composition might influence the development of resistance to chemotherapy drugs. The objective of this study was to evaluate possible expression differences in the ECM compartment of the parental human cell lines WERI-RB1 (retinoblastoma 1) and Y79 and their Etoposide resistant subclones via polymerase chain reaction (PCR). Western blot analyses were performed to analyze protein levels. To explore the influence of ECM molecules on RB cell proliferation, death, and cluster formation, WERI-RB1 and resistant WERI-ETOR cells were cultivated on Fibronectin, Laminin, Tenascin-C, and Collagen IV and analyzed via time-lapse video microscopy as well as immunocytochemistry. We revealed a significantly reduced mRNA expression of the proteoglycans Brevican, Neurocan, and Versican in resistant WERI-ETOR compared to sensitive WERI-RB1 cells. Also, for the glycoproteins α1-Laminin, Fibronectin, Tenascin-C, and Tenascin-R as well as Collagen IV, reduced expression levels were observed in WERI-ETOR. Furthermore, a downregulation was detected for the matrix metalloproteinases MMP2, MMP7, MMP9, the tissue-inhibitor of metalloproteinase TIMP2, the Integrin receptor subunits ITGA4, ITGA5 and ITGB1, and all receptor protein tyrosine phosphatase ß/ζ isoforms. Downregulation of Brevican, Collagen IV, Tenascin-R, MMP2, TIMP2, and ITGA5 was also verified in Etoposide resistant Y79 cells compared to sensitive ones. Protein levels of Tenascin-C and MMP-2 were comparable in both WERI cell lines. Interestingly, Fibronectin displayed an apoptosis-inducing effect on WERI-RB1 cells, whereas an anti-apoptotic influence was observed for Tenascin-C. Conversely, proliferation of WERI-ETOR cells was enhanced on Tenascin-C, while an anti-proliferative effect was observed on Fibronectin. In WERI-ETOR, cluster formation was decreased on the substrates Collagen IV, Fibronectin, and Tenascin-C. Collectively, we noted a different ECM mRNA expression and behavior of Etoposide resistant compared to sensitive RB cells. These findings may indicate a key role of ECM components in chemotherapy resistance formation of RB.

The Tenascin-C-Derived Peptide VSWRAPTA Promotes Neuronal Branching Via Transcellular Activation of the Focal Adhesion Kinase (FAK) and the ERK1/2 Signaling Pathway In Vitro.

The central nervous system (CNS) of mammals has a limited regeneration capacity after traumatic events, which causes chronic functional disability. The development of biomaterials aims at providing support for the regeneration process. One strategy integrates peptides that mimic functional domains of extracellular matrix (ECM) or cell adhesion molecules with synthetic polymers designed to present growth-supporting cues to the neuronal microenvironment. Thus, small peptide sequences originating from molecules of the ECM may serve as promising bio-additives, acting as artificial matricryptins to gear cellular processes. The glycoprotein tenascin-C (Tnc) is a major constituent of the ECM of the developing brain and persists in the neurogenic regions of the adult CNS. It is a multimodular glycoprotein that comprises distinct domains with neurite growth promoting and axon growth repulsing properties. In the present study, the novel peptide motif VSWRAPTA that is encoded in the neurite growth promoting 6th fibronectin type III repeat close to the alternative splice site of Tnc was tested for its effects on neuron differentiation. When this newly synthesized biomimetic peptide was added to cultures of embryonic cortical neurons it significantly promoted the outgrowth of neurites. The neuron differentiation supporting effect was thereby associated with the trans-cellular activation of the focal adhesion kinase (FAK) and the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway. Cortical neurons supplemented with the Tnc peptide displayed a dose-dependent increase in neurite outgrowth that saturated at a peptide concentration of 50 µg/ml (56.4 mMol/l). The analysis of neuron morphology revealed that neurite branching rather than fiber length was stimulated by the Tnc peptide. Therefore, we predict that the analyzed peptide motif of the 6th constitutively expressed FNIII domain of the Tnc molecule might be a major contributor for neurite outgrowth and guiding events in the native CNS microenvironment. In conclusion, the Tnc-derived VSWRAPTA peptide may represent a promising tool to spike regeneration supportive microenvironments.

The Synergistic Effect of Cationic Moieties and GRGDSF-Peptides in Hydrogels on Neural Stem Cell Behavior.

This article reports the behavior of embryonic neural stem cells on a hydrogel that combines cationic, non-specific cell adhesion motifs with glycine-arginine-glycine-aspartic acid-serine-phenylalanine (GRGDSF)-peptides as specific cell adhesion moieties. Therefore, three hydrogels are prepared by free radical polymerization that contains either a GRGDSF-peptide residue (P1), amino ethylmethacrylate as a cationic residue (P2), or a combination of both motifs (P3). For each gel, cross linker concentrations of 8 mol% is used to have a comparable gel stiffness of 8-9 kPa. The cell experiments indicate a synergistic effect of the non-specific, cationic residues, and the specific GRGDSF-peptides on embryonic neural stem cell behavior that is especially pronounced in the cell adhesion experiments by more than doubling the number of cells after 72 h when comparing P3 with P2 and is less pronounced in the proliferation and differentiation experiments.

Helional-induced activation of human olfactory receptor 2J3 promotes apoptosis and inhibits proliferation in a non-small-cell lung cancer cell line.

Studies within the last decade have localized the functional expression of olfactory receptors (ORs) to cells outside of the olfactory epithelium. In human hepatocarcinoma and prostate cancer cells, the activation of ORs by odors modulates elementary physiological processes and leads to an inhibitory effect on proliferation. Cells of the respiratory tract are in direct contact with the surrounding air, in which a myriad of volatile molecules, especially odors, are present. Non-small-cell lung cancer (NSCLC) has a high prevalence, a high mortality rate and is difficult to treat. NSCLC cells are nearly resistant to common chemotherapeutic approaches, and surgical resection provides the only possible chance of a cure for most patients. New approaches for the treatment of NSCLC are the focus of many current studies. Thus, it is of interest to characterize the functional expression of ORs in cancer cells of the lung and to investigate the impact of ORs on pathophysiological processes. In the present study, we demonstrate that the expression of OR2J3 and cytosolic Ca2+ increase via the activation of the agonist helional in the NSCLC cell line A549. We further investigated the underlying pathway. Helional triggers phoshoinositol-3 kinase (PI3K), signaling the release of intracellular Ca2+ and phosphorylation of ERK. We observed that OR2J3 activation induces apoptosis and inhibits cell proliferation and migration in long-term stimulus experiments with helional. Our study provides the first evidence of the functional expression of an OR in NSCLC cells and its putative therapeutic impact.