A series of alkenyl-substituted titanocene compounds have been supported on the mesoporous silica-based material KIT-6. The corresponding functionalised materials were completely characterised by different techniques (solid-state multinuclear NMR spectroscopy, IR spectroscopy, N2 adsorption-desorption isotherms, X-ray fluorescence and diffraction, SEM and TEM) to observe the incorporation of the titanocene derivatives on the external surface of the material KIT-6. Both the titanocene compounds and the materials were tested in vitro against a wide variety of human cancer and normal cell lines. A very high cytotoxicity of the synthesised titanocene derivatives (IC50 values in the range of those described in the literature for the most active cytotoxic titanocene compounds), with selectivity towards cancer cell lines was observed. The cytotoxic activity of the materials is the highest reported to date for titanocene-functionalised materials. In addition, higher Ti uptake (from 4 to 23% of the initial amount of Ti) of the cells treated with materials was observed with respect to those treated with "free" titanocene derivatives (which gave Ti uptake values from 0.4 to 4.6% of the initial amount of Ti). Additional experiments with the titanocene derivatives and the functionalised materials revealed that changes to the morphological and functional dynamics of apoptosis occurred when the active titanocene species were incorporated into mesoporous materials. In addition, the materials could induce programmed cell death in tumour cell populations by impairing the damaged DNA repair mechanisms and by upregulation of intrinsic and extrinsic apoptotic signalling pathways.

Silver nanoparticles (AgNPs) have been proven to have potent antibacterial properties, offering an attractive alternative to antibiotics in the treatment of several infections such as otitis media. Concerns have been raised though regarding their toxicity. There are few data regarding the toxic effects of AgNPs in cochlear cells. The aim of our study was to evaluate the effects of AgNPs of four sizes as a function of their size on HEI-OC1 cochlear cells and on HaCaT keratinocytes. The cells were treated with different concentrations of AgNPs. We evaluated silver uptake by atomic absorption spectroscopy and transmission electron microscopy (TEM), cytotoxicity with the alamarBlue test, ROS production with 2′,7′-dichloro-dihydro-fluorescein diacetate, and genotoxicity with the comet assay. Silver intracellular concentration increased proportionally with the incubation time and the size of the NPs. Silver uptake was higher in HEI-OC1 cells compared to HaCaT. While after 4 h exposure, only the 50 nm NPs were observed in both cell lines and only the 5 nm NPs were observed in the HaCaT cells, after 24 h, nanoparticles of all sizes could be visualized in both cell lines. The cells showed signs of distress: vacuolizations, autophagosomes, signs of apoptosis, or cellular debris. AgNPs of all sizes reduced viability proportionally with the concentration, HEI-OC1 cells being more affected. The toxicity of AgNPs decreased with the nanoparticle size, and ROS production was dose and size dependent, mainly in the cochlear cells. Genotoxicity assessed by comet assay revealed a higher level of DNA lesions in HEI-OC1 cells after treatment with small-sized AgNPs. The perspective of using AgNPs in the treatment of otitis media, although very attractive, must be regarded with caution: cochlear cells proved to be more sensitive to the toxic effect of AgNPs compared to other cell lines. Potential treatments must be tailored specifically, choosing NPs with minimum toxicity towards auditory cells.

The major green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) has been shown to exhibit antitumour activities in several tumour models. One of the possible mechanisms by which EGCG can inhibit cancer progression is through the modulation of angiogenesis signalling cascade. The tumour cells’ ability to tightly adhere to endothelium is a very important process in the metastatic process, because once disseminated into the bloodstream the tumour cells must re-establish adhesive connections to endothelium in order to extravasate into the target tissues. In this study, we investigated the anti-angiogenic effects of EGCG treatment (10 μM) on human cervical tumour cells (HeLa) by evaluating the changes in the expression pattern of 84 genes known to be involved in the angiogenesis process. Transcriptional analysis revealed 11 genes to be differentially expressed and was further validated by measuring the induced biological effects. Our results show that EGCG treatment not only leads to the down-regulation of genes involved in the stimulation of proliferation, adhesion and motility as well as invasion processes, but also to the up-regulation of several genes known to have antagonist effects. We observed reduced proliferation rates, adhesion and spreading ability as well as invasiveness of HeLa tumour cells upon treatment, which suggest that EGCG might be an important anti-angiogenic therapeutic approach in cervical cancers.

Gold nanoparticles (GNPs) were obtained by green synthesis with an extract fromCornus masfruits (GNPs-CM), characterized by several methods, and their biologic effects were evaluated on two cell lines: HaCaT, normal keratinocytes, and A431, epidermoid carcinoma. GNPs were spherical with sizes between 2 and 24 nm. Their optical spectra had a dominant plasmonic band centered at 525 nm; zeta potential distribution was narrow, centered at −19.7 mV, and the mean hydrodynamic diameter was 58 nm. GNPs were visualized in both cell types entering the cells by endocytosis. The amount of gold uptaken by the cells was dose and time dependent. The intracellular concentration of Au ions was higher in HaCaT compared to A431 cells. The toxicity of GNPs-CM was dose dependent being significant only when the highest concentrations were employed. A431 cells were less affected compared to HaCaT cells, but the difference was not statistically significant. ROS production was not significant, except in HaCaT cells at the highest concentration. The comet assay revealed no significant supplementary DNA lesions, while the secretion of inflammatory cytokines was modulated by the presence of GNPs only when the cells were additionally irradiated with UVB. These results recommend GNPs-CM for further testing and possible dermatological applications.

BackgroundCancer cells frequently adopt cellular and molecular alterations and acquire resistance to cytostatic drugs. Chemotherapy with oxaliplatin is among the leading treatments for colorectal cancer with a response rate of 50%, inducing intrastrand cross-links on the DNA. Despite of this drug’s efficiency, resistance develops in nearly all metastatic patients. Chemoresistance being of crucial importance for the drug’s clinical efficiency this study aimed to contribute to the identification and description of some cellular and molecular alterations induced by prolonged oxaliplatin therapy. Resistance to oxaliplatin was induced in Colo320 (Colo320R) and HT-29 (HT-29R) colorectal adenocarcinoma cell lines by exposing the cells to increasing concentrations of the drug. Alterations in morphology, cytotoxicity, DNA cross-links formation and gene expression profiles were assessed in the parental and resistant variants with microscopy, MTT, alkaline comet and pangenomic microarray assays, respectively.ResultsMorphology analysis revealed epithelial-to-mesenchymal transition in the resistant vs parental cells suggesting alterations of the cells’ adhesion complexes, through which they acquire increased invasiveness and adherence. Cytotoxicity measurements demonstrated resistance to oxaliplatin in both cell lines; Colo320 being more sensitive than HT-29 to this drug (P < 0.001). The treatment with oxaliplatin caused major DNA cross-links in both parental cell lines; in Colo320R small amounts of DNA cross-links were still detectable, while in HT-29R not. We identified 441 differentially expressed genes in Colo320R and 613 in HT-29R as compared to their parental counterparts (at least 1.5 -fold up- or down- regulation, p < 0.05). More disrupted functions and pathways were detected in HT-29R cell line than in Colo320R, involving genes responsible for apoptosis inhibition, cellular proliferation and epithelial-to-mesenchymal transition. Several upstream regulators were detected as activated in HT-29R cell line, but not in Colo320R.ConclusionsOur findings revealed a more resistant phenotype in HT-29R as compared to Colo320R and different cellular and molecular chemoresistance patterns induced by prolonged treatment with oxaliplatin in cell lines with identical origins (colorectal adenocarcinomas).

BackgroundThe development of novel biomaterials able to control cell activities and direct their fate is warranted for engineering functional bone tissues. Adding bioactive materials can improve new bone formation and better osseointegration. Three types of titanium (Ti) implants were tested for in vitro biocompatibility in this comparative study: Ti6Al7Nb implants with 25% total porosity used as controls, implants infiltrated using a sol–gel method with hydroxyapatite (Ti HA) and silicatitanate (Ti SiO2). The behavior of human osteoblasts was observed in terms of adhesion, cell growth and differentiation.ResultsThe two coating methods have provided different morphological and chemical properties (SEM and EDX analysis). Cell attachment in the first hour was slower on the Ti HA scaffolds when compared to Ti SiO2 and porous uncoated Ti implants. The Alamar blue test and the assessment of total protein content uncovered a peak of metabolic activity at day 8–9 with an advantage for Ti SiO2 implants. Osteoblast differentiation and de novo mineralization, evaluated by osteopontin (OP) expression (ELISA and immnocytochemistry), alkaline phosphatase (ALP) activity, calcium deposition (alizarin red), collagen synthesis (SIRCOL test and immnocytochemical staining) and osteocalcin (OC) expression, highlighted the higher osteoconductive ability of Ti HA implants. Higher soluble collagen levels were found for cells cultured in simple osteogenic differentiation medium on control Ti and Ti SiO2 implants. Osteocalcin (OC), a marker of terminal osteoblastic differentiation, was most strongly expressed in osteoblasts cultivated on Ti SiO2 implants.ConclusionsThe behavior of osteoblasts depends on the type of implant and culture conditions. Ti SiO2 scaffolds sustain osteoblast adhesion and promote differentiation with increased collagen and non-collagenic proteins (OP and OC) production. Ti HA implants have a lower ability to induce cell adhesion and proliferation but an increased capacity to induce early mineralization. Addition of growth factors BMP-2 and TGFβ1 in differentiation medium did not improve the mineralization process. Both types of infiltrates have their advantages and limitations, which can be exploited depending on local conditions of bone lesions that have to be repaired. These limitations can also be offset through methods of functionalization with biomolecules involved in osteogenesis.

Objectives: Cone-beam CT (CBCT) is a medical imaging technique used in dental medicine. However, there are no conclusive data available indicating that exposure to X-ray doses used by CBCT are harmless. We aim, for the first time, to characterize the potential age-dependent cellular and subcellular effects related to exposure to CBCT imaging. Current objective is to describe and validate the protocol for characterization of cellular and subcellular changes after diagnostic CBCT. Methods: Development and validation of a dedicated two-part protocol: 1) assessing DNA double strand breaks (DSBs) in buccal mucosal (BM) cells and 2) oxidative stress measurements in saliva samples. BM cells and saliva samples are collected prior to and 0.5 h after CBCT examination. BM cells are also collected 24 h after CBCT examination. DNA DSBs are monitored in BM cells via immunocytochemical staining for γH2AX and 53BP1. 8-oxo-7,8-dihydro-2’-deoxyguanosine (8-oxo-dG) and total antioxidant capacity are measured in saliva to assess oxidative damage. Results: Validation experiments show that sufficient BM cells are collected (97.1 ± 1.4 %) and that γH2AX/53BP1 foci can be detected before and after CBCT examination. Collection and analysis of saliva samples, either sham exposed or exposed to IR, show that changes in 8-oxo-dG and total antioxidant capacity can be detected in saliva samples after CBCT examination. Conclusion: The DIMITRA Research Group presents a two-part protocol to analyze potential age-related biological differences following CBCT examinations. This protocol was validated for collecting BM cells and saliva and for analyzing these samples for DNA DSBs and oxidative stress markers, respectively.

Platinum-based chemotherapeutic agents are considered among the most potent anticancer drugs used in the treatment of human tumors. Cisplatin is efficient in the treatment of testicular, ovarian, bladder, and head and neck carcinomas, although its use is limited by severe nephrotoxicity and ototoxicity and resistance. Oxaliplatin has consistently exerted antitumor activity in colon, ovarian, and lung cancers and shown less toxicity than its analogue. Given that most of the literature data are contradictory with respect to the cytotoxicity of these drugs and DNA adduct formation, the present study aimed to determine some of the potential underlying mechanisms in view of their cellular uptakes. We evaluated the cytotoxicity, DNA cross-link formation, and cellular uptake of cisplatin and oxaliplatin in Colo320, HT-29, and Caco-2 colorectal adenocarcinoma cell lines. Our results showed higher cytotoxicity of oxaliplatin in Colo320 (P<0.05) and HT-29 cell lines and of cisplatin in Caco-2 (P<0.05). Oxaliplatin induced more DNA cross-links than cisplatin in a dose-dependent manner in Colo320 cells (P<0.0001); in HT-29 and Caco-2 cells, the induction of DNA damage was not dose dependent. Multiple accumulation of cisplatin versus oxaliplatin occurred in all the cell types, doses, and time points we tested. Oxaliplatin showed more potent biological activities versus cisplatin in terms of a significantly lower cellular uptake. In addition to their analogous mechanisms of action, these drugs might activate different signal transduction pathways, ultimately leading to apoptotic DNA fragmentation and cell death. DNA damage, although perhaps the most important, represents only one aspect of the multiple effects of platinum drugs.