Substrate viscosity impairs temozolomide-mediated inhibition of glioblastoma cells' growth.

BACKGROUND: The mechanical state of the extracellular environment of the brain cells considerably affects their phenotype during the development of central nervous system (CNS) pathologies, and when the cells respond to drugs. The reports on the evaluation of the viscoelastic properties of different brain tumors have shown that both tissue stiffness and viscosity can be altered during cancer development. Although a compelling number of reports established the role of substrate stiffness on the proliferation, motility, and drug sensitivity of brain cancer cells, there is a lack of parallel data in terms of alterations in substrate viscosity. METHODS: Based on viscoelasticity measurements of rat brain samples using strain rheometry, polyacrylamide (PAA) hydrogels mimicking elastic and viscous parameters of the tissues were prepared. Optical microscopy and flow cytometry were employed to assess the differences in glioblastoma cells morphology, proliferation, and cytotoxicity of anticancer drug temozolomide (TMZ) due to increased substrate viscosity. RESULTS: Our results indicate that changes in substrate viscosity affect the proliferation of untreated glioma cells to a lesser extent, but have a significant impact on the apoptosis-associated depolarization of mitochondria and level of DNA fragmentation. This suggests that viscosity sensing and stiffness sensing machinery can activate different signaling pathways in glioma cells. CONCLUSION: Collected data indicate that viscosity should be considered an important parameter in in vitro polymer-based cell culture systems used for drug screening.

Mechanical Properties of the Extracellular Environment of Human Brain Cells Drive the Effectiveness of Drugs in Fighting Central Nervous System Cancers.

The evaluation of nanomechanical properties of tissues in health and disease is of increasing interest to scientists. It has been confirmed that these properties, determined in part by the composition of the extracellular matrix, significantly affect tissue physiology and the biological behavior of cells, mainly in terms of their adhesion, mobility, or ability to mutate. Importantly, pathophysiological changes that determine disease development within the tissue usually result in significant changes in tissue mechanics that might potentially affect the drug efficacy, which is important from the perspective of development of new therapeutics, since most of the currently used in vitro experimental models for drug testing do not account for these properties. Here, we provide a summary of the current understanding of how the mechanical properties of brain tissue change in pathological conditions, and how the activity of the therapeutic agents is linked to this mechanical state.

N-Acetyl-Cysteine Increases Activity of Peanut-Shaped Gold Nanoparticles Against Biofilms Formed by Clinical Strains of Pseudomonas aeruginosa Isolated from Sputum of Cystic Fibrosis Patients.

Background: Extracellular polymeric substances (EPS) produced by bacteria, as they form a biofilm, determine the stability and viscoelastic properties of biofilms and prevent antibiotics from penetrating this multicellular structure. To date, studies demonstrated that an appropriate optimization of the chemistry and morphology of nanotherapeutics might provide a favorable approach to control their interaction with EPS and/or diffusion within the biofilm matrix. Targeting the biofilms' EPS, which in certain conditions can adopt liquid crystal structure, was demonstrated to improve the anti-biofilm activity of antibiotics and nanoparticles. A similar effect is achievable by interfering EPS' production by mucoactive agents, such as N-acetyl-cysteine (NAC). In our previous study, we demonstrated the nanogram efficiency of non-spherical gold nanoparticles, which due to their physicochemical features, particularly morphology, were noted to be superior in antimicrobial activity compared to their spherical-shaped counterparts. Methods: To explore the importance of EPS matrix modulation in achieving a suitable efficiency of peanut-shaped gold nanoparticles (AuP NPs) against biofilms produced by Pseudomonas aeruginosa strains isolated from cystic fibrosis patients, fluorescence microscopy, as well as resazurin staining were employed. Rheological parameters of AuP NPs-treated biofilms were investigated by rotational and creep-recovery tests using a rheometer in a plate-plate arrangement. Results: We demonstrated that tested nanoparticles significantly inhibit the growth of mono- and mixed-species biofilms, particularly when combined with NAC. Notably, gold nanopeanuts were shown to decrease the viscosity and increase the creep compliance of Pseudomonas biofilm, similarly to EPS-targeting NAC. Synergistic activity of AuP NPs with tobramycin was also observed, and the AuP NPs were able to eradicate bacteria within biofilms formed by tobramycin-resistant isolates. Conclusion: We propose that peanut-shaped gold nanoparticles should be considered as a potent therapeutic agent against Pseudomonas biofilms.

Inhomogeneity of stiffness and density of the extracellular matrix within the leukoplakia of human oral mucosa as potential physicochemical factors leading to carcinogenesis.

Oral leukoplakia is a clinical term relating to various morphological lesions, including squamous cell hyperplasia, dysplasia and carcinoma. Leukoplakia morphologically manifested as hyperplasia with epithelial dysplasia is clinically treated as precancerous condition. Nevertheless, there is a lack of good markers indicating the transformation of premalignancies towards cancer. A better understanding of the mechanical environment within the tissues where tumors grow might be beneficial for the development of prevention, diagnostic, and treatment methods in cancer management. Atomic force microscopy (AFM) and immunohistology techniques were used to assess changes in the stiffness and morphology of oral mucosa and leukoplakia samples at different stages of their progression towards cancer. The Young's moduli of the tested leukoplakia samples were significantly higher than those of the surrounding mucus. Robust inhomogeneity of stiffness within leukoplakia samples, reflecting an increase in regeneration and collagen accumulation (increasing density) in the extracellular matrix (ECM), was observed. Within the histologically confirmed cancer samples, Young's moduli were significantly lower than those within the precancerous ones. Inhomogeneous stiffness within leukoplakia might act as "a mechanoagonist" that promotes oncogenesis. In contrast, cancer growth might require the reorganization of tissue structure to create a microenvironment with lower and homogenous stiffness. The immunohistology data collected here indicates that changes in tissue stiffness are achieved by increasing cell/ECM density. The recognition of new markers of premalignancy will aid in the development of new therapies and will expand the diagnostic methods.

Nanomechanics and Histopathology as Diagnostic Tools to Characterize Freshly Removed Human Brain Tumors.

BACKGROUND: The tissue-mechanics environment plays a crucial role in human brain physiological development and the pathogenesis of different diseases, especially cancer. Assessment of alterations in brain mechanical  properties during cancer progression might provide important information about possible tissue abnormalities with clinical relevance. METHODS: With atomic force microscopy (AFM), the stiffness of freshly removed human brain tumor tissue was determined on various regions of the sample and compared to the stiffness of healthy human brain tissue that was removed during neurosurgery to gain access to tumor mass. An advantage of indentation measurement using AFM is the small volume of tissue required and high resolution at the single-cell level. RESULTS: Our results showed great heterogeneity of stiffness within metastatic cancer or primary high-grade gliomas compared to healthy tissue. That effect was not clearly visible in lower-grade tumors like meningioma. CONCLUSION: Collected data indicate that AFM might serve as a diagnostic tool in the assessment of human brain tissue stiffness in the process of recognizing tumors.

Bacteria Residing at Root Canals Can Induce Cell Proliferation and Alter the Mechanical Properties of Gingival and Cancer Cells.

Understanding the importance of oral microbiota in human health and disease also leads to an expansion of the knowledge on functional, metabolic, and molecular alterations directly contributing to oral and systemic pathologies. To date, a compelling number of studies have documented the crucial role of some oral cavity-occurring microbes in the initiation and progression of cancers. Although this effect was noted primarily for Fusobacterium spp., the potential impact of other oral microbes is also worthy of investigation. In this study, we aimed to assess the effect of Enterococcus faecalis, Actinomyces odontolyticus, and Propionibacterium acnes on the proliferation capability and mechanical features of gingival cells and cell lines derived from lung, breast, and ovarian cancers. For this purpose, we incubated selected cell lines with heat-inactivated bacteria and supernatants collected from biofilms, cultured in both anaerobic and aerobic conditions, in the presence of surgically removed teeth and human saliva. The effect of oral bacteria on cell population growth is variable, with the highest growth-promoting abilities observed for E. faecalis in relation to human primary gingival fibroblasts (HGF) and lung cancer A549 cells, and P. acnes in relation to breast cancer MCF-7 and ovarian cancer SKOV-3 cells. Notably, this effect seems to depend on a delicate balance between the pro-stimulatory and toxic effects of bacterial-derived products. Regardless of the diverse effect of bacterial products on cellular proliferation capability, we observed significant alterations in stiffness of gingival and lung cancer cells stimulated with E. faecalis bacteria and corresponding biofilm supernatants, suggesting a novel molecular mechanism involved in the pathogenesis of diseases in oral cavities and tooth tissues. Accordingly, it is proposed that analysis of cancerogenic features of oral cavity bacteria should be multivariable and should include investigation of potential alterations in cell mechanical properties. These findings corroborate the important role of oral hygiene and root canal treatment to assure the healthy stage of oral microbiota.

Tissue Rheology as a Possible Complementary Procedure to Advance Histological Diagnosis of Colon Cancer.

In recent years, rheological measurements of cells and tissues at physiological and pathological stages have become an essential method to determine how forces and changes in mechanical properties contribute to disease development and progression, but there is no standardization of this procedure so far. In this study, we evaluate the potential of nanoscale atomic force microscopy (AFM) and macroscopic shear rheometry to assess the mechanical properties of healthy and cancerous human colon tissues. The direct comparison of tissue mechanical behavior under uniaxial and shear deformation shows that cancerous tissues not only are stiffer compared to healthy tissue but also respond differently when shear and compressive stresses are applied. These results suggest that rheological parameters can be useful measures of colon cancer mechanopathology. Additionally, we extend the list of biological materials exhibiting compressional stiffening and shear weakening effects to human colon tumors. These mechanical responses might be promising mechanomarkers and become part of the new procedures in colon cancer diagnosis. Enrichment of histopathological grading with rheological assessment of tissue mechanical properties will potentially allow more accurate colon cancer diagnosis and improve prognosis.

Biofilm Growth Causes Damage to Silicone Voice Prostheses in Patients after Surgical Treatment of Locally Advanced Laryngeal Cancer.

Voice prosthesis implantation with the creation of a tracheoesophageal fistula is the gold standard procedure for voice rehabilitation in patients after a total laryngectomy. All patients implanted with a voice prosthesis (VP) have biofilms of fungi and bacteria grow on their surface. Biofilm colonization is one of the main reasons for VP degradation that can lead to VP dysfunction, which increases the high risk of pneumonia. In a 20-month evaluation period, 129 cases of prostheses after replacement procedures were investigated. Microbiological examination of the biofilms revealed that there were four of the most common fungi species (Candida spp.) and a large variety of bacterial species present. We studied the relationship between the time of proper function of Provox VP, the microorganism composition of the biofilm present on it, and the degradation level of the silicone material. Evaluation of the surface of the removed VP using an atomic force microscope (AFM) has demonstrated that biofilm growth might drastically change the silicone's mechanical properties. Changes in silicone stiffness and thermal properties might contribute to the failure of VP function. Our data can serve in future studies for the development of methods to prevent or inhibit biofilm formation on the VP surface that would translate to an increase in their durability and safety.

Recombinant Human Plasma Gelsolin Stimulates Phagocytosis while Diminishing Excessive Inflammatory Responses in Mice with Pseudomonas aeruginosa Sepsis.

Plasma gelsolin (pGSN) is a highly conserved abundant circulating protein, characterized by diverse immunomodulatory activities including macrophage activation and the ability to neutralize pro-inflammatory molecules produced by the host and pathogen. Using a murine model of Gram-negative sepsis initiated by the peritoneal instillation of Pseudomonas aeruginosa Xen 5, we observed a decrease in the tissue uptake of IRDye®800CW 2-deoxyglucose, an indicator of inflammation, and a decrease in bacterial growth from ascitic fluid in mice treated with intravenous recombinant human plasma gelsolin (pGSN) compared to the control vehicle. Pretreatment of the murine macrophage line RAW264.7 with pGSN, followed by addition of Pseudomonas aeruginosa Xen 5, resulted in a dose-dependent increase in the proportion of macrophages with internalized bacteria. This increased uptake was less pronounced when cells were pretreated with pGSN and then centrifuged to remove unbound pGSN before addition of bacteria to macrophages. These observations suggest that recombinant plasma gelsolin can modulate the inflammatory response while at the same time augmenting host antibacterial activity.

Inhibition of inflammatory response in human keratinocytes by magnetic nanoparticles functionalized with PBP10 peptide derived from the PIP2-binding site of human plasma gelsolin.

BACKGROUND: Human plasma gelsolin (pGSN) is a multifunctional actin-binding protein involved in a variety of biological processes, including neutralization of pro-inflammatory molecules such as lipopolysaccharide (LPS) and lipoteichoic acid (LTA) and modulation of host inflammatory response. It was found that PBP10, a synthetic rhodamine B-conjugated peptide, based on the phosphoinositide-binding site of pGSN, exerts bactericidal activity against Gram-positive and Gram-negative bacteria, interacts specifically with LPS and LTA, and limits microbial-induced inflammatory effects. The therapeutic efficiency of PBP10 when immobilized on the surface of iron oxide-based magnetic nanoparticles was not evaluated, to date. RESULTS: Using the human keratinocyte cell line HaCaT stimulated by bacterially-derived LPS and LTA as an in vitro model of bacterial infection, we examined the anti-inflammatory effects of nanosystems consisting of iron oxide-based magnetic nanoparticles with aminosilane (MNP@NH2) or gold shells (MNP@Au) functionalized by a set of peptides, derived from the phosphatidylinositol 4,5-bisphosphate (PIP2)-binding site of the human plasma protein gelsolin, which also binds LPS and LTA. Our results indicate that these nanosystems can kill both Gram-positive and Gram-negative bacteria and limit the production of inflammatory mediators, including nitric oxide (NO), reactive oxygen species (ROS), and interleukin-8 (IL-8) in the response to heat-killed microbes or extracted bacterial cell wall components. The nanoparticles possess the potential to improve therapeutic efficacy and are characterized by lower toxicity and improved hemocompatibility when compared to free peptides. Atomic force microscopy (AFM) showed that these PBP10-based nanosystems prevented changes in nanomechanical properties of cells that were otherwise stimulated by LPS. CONCLUSIONS: Neutralization of endotoxemia-mediated cellular effects by gelsolin-derived peptides and PBP10-containing nanosystems might be considered as potent therapeutic agents in the improved therapy of bacterial infections and microbial-induced inflammation.

Ceragenin CSA-13 as free molecules and attached to magnetic nanoparticle surfaces induce caspase-dependent apoptosis in human breast cancer cells via disruption of cell oxidative balance.

Natural antimicrobial peptides and ceragenins, as non-peptide amphipathic mimics, have been proposed as anti-cancer agents. To date, it has been confirmed that cathelicidin LL-37 and ceragenin CSA-13, both in free form and immobilized on the surface of magnetic nanoparticles (MNP@LL-37, MNP@CSA-13) induce apoptosis in colon cancer cells. Nevertheless, the question remains whether ceragenins, as synthetic analogs of LL-37 peptide and mimicking a number of its properties, act as antineoplastic agents in breast cancer cells, where LL-37 peptide stimulates oncogenesis. Considering potential anticancer activity, we determined whether CSA-13 and MNP@CSA-13 might be effective against breast cancer cells. Our study provides evidence that both CSA-13 and MNP@CSA-13 decreased viability and inhibit proliferation of MCF-7 and MDA-MB-231 cells despite the protumorigenic properties of LL-37 peptide. Flow cytometry-based analyses revealed that ceragenin treatment results in increases in dead and PI-negative/low-viability cells, which was associated with glutathione (GSH) depletion and increased reactive oxygen species (ROS) generation followed by mitochondrial membrane depolarization, caspase activation, and DNA fragmentation. These findings demonstrate that both CSA-13 and MNP@CSA-13 cause disruption of the oxidative balance of cancer cells. This novel mechanism of ceragenin-mediated eradication of cancer cells suggest that these agents may be developed as a possible treatment of breast cancer.