Zeaxanthin-Rich Extract from Superfood Lycium barbarum Selectively Modulates the Cellular Adhesion and MAPK Signaling in Melanoma versus Normal Skin Cells In Vitro.

The concern for implementing bioactive nutraceuticals in antioxidant-related therapies is of great importance for skin homeostasis in benign or malignant diseases. In order to elucidate some novel insights of Lycium barbarum (Goji berry) activity on skin cells, the present study focused on its active compound zeaxanthin. By targeting the stemness markers CD44 and CD105, with deep implications in skin oxidative stress mechanisms, we revealed, for the first time, selectivity in zeaxanthin activity. When applied in vitro on BJ human fibroblast cell line versus the A375 malignant melanoma cells, despite the moderate cytotoxicity, the zeaxanthin-rich extracts 1 and 2 were able to downregulate significantly the CD44 and CD105 membrane expression and extracellular secretion in A375, and to upregulate them in BJ cells. At mechanistic level, the present study is the first to demonstrate that the zeaxanthin-rich Goji extracts are able to influence selectively the mitogen-activated protein kinases (MAPK): ERK, JNK and p38 in normal BJ versus tumor-derived A375 skin cells. These results point out towards the applications of zeaxanthin from L. barbarum as a cytoprotective agent in normal skin and raises questions about its use as an antitumor prodrug alone or in combination with standard therapy.

Synthesis, structural characterization and biological activity of novel Knoevenagel condensates on DLD-1 human colon carcinoma.

Biologically active Knoevenagel condensates (1-14) of diarylheptanoids: 1,7-bis(3-methoxy-4-hydroxyphenyl)hepta-1,7-diene-3,5-dione and 1,7-bis(3-ethoxy-4-hydroxyphenyl)hepta-1,7-diene-3,5-dione, were synthesized and structurally characterized. Compounds 1-14 exhibited cytotoxicity against colon carcinoma cells, and their antiproliferative effect was associated with a significant decrease of multidrug resistance proteins. One of the underlying mechanisms of these effects is the reduction of intracellular and extracellular SOD enzymes by compounds 1, 12 and 14, which render the tumor cells more vulnerable to oxidative stress.

Novel Palladium(II) Complexes that Influence Prominin-1/CD133 Expression and Stem Cell Factor Release in Tumor Cells.

New Pd(II) complexes of 1,7-bis(2-methoxyphenyl)hepta-1,6-diene-3,5-dione were synthesized and structurally characterized. The complexes were tested in vitro on human colon and hepatic carcinoma cell lines, normal hepatic cells and hematopoietic progenitor cells. Biological tests proved that Pd(II) complexes 1 and 2 (containing a curcumin derivative) exhibit a strong in vitro antitumor effect against the cells derived from human colorectal carcinoma and the hepatic metastasis of a colorectal carcinoma. Complex 1 has an outstanding inhibitory effect against BRAF-mutant colon carcinoma and hepatocarcinoma cell growth; 1 and 2 are both more active than the free ligand and have the capacity to trigger early apoptotic processes. By flow cytometric measurements, an important decrease of prominin-1 (CD133) molecule expression on tumor cells membrane was identified in cell populations subjected to 1 and 2. Quantitative immune enzymatic assay proved restrictions in stem cell factor (SCF) release by treated tumor cells. Although less cytotoxic, the free ligand inhibits the surface marker CD133 expression in hepatocarcinoma cells, and in HT-29 colon carcinoma. The new synthesized Pd(II) complexes 1 and 2 exhibit an important potential through their selective cytotoxic activity and by targeting the stem-like tumor cell populations, which leads to the tumor growth arrest and prevention of metastasis.

Platinum derivatives: a multidisciplinary approach.

Cancer is one of the most difficult diseases to be treated. The particularities regarding the tumors' occurrence mechanism, their evolution under chemotherapy, disease-free interval, but also the increasing number of patients make cancer an intensively studied health domain. Although introduced in therapy since the early 80s, platinum derivatives play an essential role in anticancer therapy. Their use in therapy resulted in improving the patient quality of life and prolonging disease-free interval, which makes them still a benchmark for other anticancer compounds. However, adverse reactions and allergic reactions are a major impediment in therapy with platinum derivatives. This paper summarizes data about platinum derivatives through a multidisciplinary approach, starting from a chemical point of view and on to their mechanism of action, mechanism of cellular resistance, predictive factors for the outcome of chemotherapy such as micro RNAs (miRNAs), tumor suppressor protein p53, and the excision repair cross-complementing 1 protein (ERCC1).

Anti-cancer applications of titanocene-functionalised nanostructured systems: an insight into cell death mechanisms.

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.

Investigation of Epilobium hirsutum L. Optimized Extract's Anti-Inflammatory and Antitumor Potential.

Epilobium hirsutum L., commonly known as hairy willowherb, is a perennial herbaceous plant native to Europe and Asia. In Romania, the Epilobium genus includes 17 species that are used in folk medicine for various purposes. This study aimed to investigate the anti-inflammatory and antitumor potential of the optimized extract of Epilobium hirsutum (EH) in animal models. The first study investigated the anti-inflammatory properties of EH optimized extract and the model used was carrageenan-induced paw inflammation. Wistar rats were divided into three groups: negative control, positive control treated with indomethacin, and a group treated with the extract. Oxidative stress markers, cytokine levels, and protein expressions were assessed. The extract demonstrated anti-inflammatory properties comparable to those of the control group. In the second study, the antitumor effects of the extract were assessed using the tumor model of Ehrlich ascites carcinoma. Swiss albino mice with Ehrlich ascites were divided into four groups: negative, positive treated with cyclophosphamide (Cph), Group 3 treated with Cph and EH optimized extract, and Group 4 treated with extract alone. Samples from the ascites fluid, liver, and heart were analyzed to evaluate oxidative stress, inflammation, and cancer markers. The extract showed a reduction in tumor-associated inflammation and oxidative stress. Overall, the EH optimized extract exhibited promising anti-inflammatory and antitumor effects in the animal models studied. These findings suggest its potential as a natural adjuvant therapeutic agent for addressing inflammation and oxidative stress induced by different pathologies.

Oxaliplatin induces different cellular and molecular chemoresistance patterns in colorectal cancer cell lines of identical origins.

BACKGROUND: Cancer 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. RESULTS: Morphology 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. CONCLUSIONS: Our 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).

An In Vitro and In Vivo Assessment of Antitumor Activity of Extracts Derived from Three Well-Known Plant Species.

One of the objectives of this study consists of the assessment of the antitumor activity of several extracts from three selected plant species: Xanthium spinosum L., Trifolium pratense L., and Coffea arabica L. and also a comparative study of this biological activity, with the aim of establishing a superior herbal extract for antitumor benefits. The phytochemical profile of the extracts was established by HPLC-MS analysis. Further, the selected extracts were screened in vitro for their antitumor activity and antioxidant potential on two cancer cell lines: A549-human lung adenocarcinoma and T47D-KBluc-human breast carcinoma and on normal cells. One extract per plant was selected for in vivo assessment of antitumor activity in an Ehrlich ascites mouse model. The extracts presented high content of antitumor compounds such as caffeoylquinic acids in the case of X. spinosum L. (7.22 µg/mL-xanthatin, 4.611 µg/mL-4-O-caffeoylquinic acid) and green coffee beans (10.008 µg/mL-cafestol, 265.507 µg/mL-4-O-caffeoylquinic acid), as well as isoflavones in the case of T. pratense L. (6806.60 ng/mL-ononin, 102.78 µg/mL-biochanin A). Concerning the in vitro results, the X. spinosum L. extracts presented the strongest anticancerous and antioxidant effects. In vivo, ascites cell viability decreased after T. pratense L. and green coffee bean extracts administration, whereas the oxidative stress reduction potential was important in tumor samples after T. pratense L. Cell viability was also decreased after administration of cyclophosphamide associated with X. spinosum L. and T. pratense L. extracts, respectively. These results suggested that T. pratense L. or X. spinosum L. extracts in combination with chemotherapy can induce lipid peroxidation in tumor cells and decrease the tumor viability especially, T. pratense L. extract.

Early transcriptional pattern of angiogenesis induced by EGCG treatment in cervical tumour 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.

Superior cytotoxicity and DNA cross-link induction by oxaliplatin versus cisplatin at lower cellular uptake in colorectal cancer cell lines.

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.

A comparative study of genotoxicity endpoints for women exposed to different levels of indoor radon concentrations.

BACKGROUND AND PURPOSE: Radon and its radioactive progenies are the most important source of ionizing radiation of natural origin, being classified as a Group 1 carcinogen. The aim of this study is to investigate the genotoxic effects of a wide range of indoor radon concentrations, as well as the kinetics of the process of repairing DNA-induced lesions by a challenging dose of gamma irradiation. MATERIAL AND METHODS: Female subjects residing in the Baita-Stei radon priority area were selected as the exposed group. The reference group was comprised of women from the same county (Bihor), but located in an area with an average indoor radon concentration typical of the county from which they were taken. Radon concentration values of 300 Bq/m3 and 148 Bq/m3, respectively, were chosen as a threshold in order to capture the impact of radon exposure between the groups. The alkaline comet assay was used in order to measure the DNA damage, as well as the repair kinetics at 2 and 24 h after 2 Gy challenging doses of gamma irradiation using peripheral blood lymphocytes. From the serum of the subjects, the oxidative damage by 8-hydroxydeoxyguanosine as well as the PARP induction was evaluated. The chromosomal aberrations were evaluated using the Cytokinesis Block MicroNucleus Assay. RESULTS: A statistically significant increase was observed in terms of DNA-induced lesions assessed by comet assay for the exposed group compared to the reference group. A positive correlation was obtained between DNA damage and the annual effective dose, respectively with the radon progenies concentrations. A statistically significant difference was also observed for the frequency of the micronuclei between the exposed - reference groups. Significantly faster repair kinetics of DNA-induced lesions was recorded for the first 2 h after gamma irradiation in the reference group compared to the exposed group. Using the threshold of 300 Bq/m3 for radon concentration, faster kinetics of DNA damage repair for people exposed to low radon concentrations, compared to those exposed to higher concentrations for the second phase of DNA repair kinetics was observed. CONCLUSION: An increased radiosensitivity of lymphocytes, as well as slower repair kinetics, may be associated with exposure to higher indoor radon concentrations.

Assessment of cytotoxicity, apoptosis and DNA damages in Colo320 colorectal cancer cells selected for oxaliplatin resistance.

Despite the notable efficacy of oxaliplatin in the treatment of colorectal cancers, the metastatic tumours ultimately become resistant to the drug. This study investigated whether the oxaliplatin-resistant cells display different behaviour to this drug versus the sensitive cells and if this difference may be further exploited into the clinical treatments improvement. In order to establish a stable cell line resistant to oxaliplatin, a human colorectal cancer cell line (Colo320) was exposed to increasing doses of the drug up to the clinically relevant plasma concentration. Four cell groups with different levels of chemoresistance were subjected to additional doses of oxaliplatin, and their cytotoxicity, apoptosis and DNA damage production were assessed. Cells selected for resistance to oxaliplatin reacted differently to the application of additional doses of the drug, displaying lower toxicity and cellular death and fewer DNA cross-links formation, in accordance with the extent of the oxaliplatin pretreatments. As the cross-links formation by oxaliplatin being the main cause for cytotoxicity of this drug and a correlation between cytotoxicity and clinical outcome being shown repeatedly, we consider that the evaluation of oxaliplatin-induced cytotoxicity, apoptosis and DNA damage could be a valuable tool to assess the tumour cells sensitivity and thus to predict the response to chemotherapy.

Influence of palm oil on doxorubicin induced cytotoxicity in normal and tumor cell cultures.

The vitamin E of palm oil, unlike most other vegetal fats, consists largely of tocotrienols (TT), products previously reported as having antioxidant and tumor-inhibitory properties. A tocotrienols containing palm oil, in the form of liposomes entrapping dosages of 0.5-0.05 microgTT/mL, was studied in combined treatments with doxorubicin (30 min before drug administration). The IC(50) values of doxorubicin, at 24 h, showed that its cytotoxic effects were decreased by palm oil, in a dose effect relationship (p < 0.01, ANOVA), in both normal (Hfl-1, Huvec) and tumor (HepG2, Mls) cells. These results demonstrated an unselective protective activity of tocotrienols, in vitro, on some normal and tumor cultured cells treated with doxorubicin.

Enhanced chemoresistance and tumor sphere formation as a laboratory model for peritoneal micrometastasis in epithelial ovarian cancer.

BACKGROUND AND PURPOSE: Ovarian cancers are composed of heterogeneous cell populations, including highly proliferative immature precursors and differentiated cells that may belong to different lineages. The main reason why epithelial ovarian cancer is difficult to treat is the unusual mechanism of dissemination that involves local invasion of pelvic and abdominal organs. But, unlike many other carcinomas, initial dissemination rarely requires blood or lymph vessels. Because it has been proven that aggregates of malignant cells within the ascites of patients diagnosed with ovarian cancer represent an impediment to cure such cancers, in the present study we adopted suspension culture combined with anti-cancer regimens as a laboratory strategy for research of the initial process of peritoneal micrometastasis. EXPERIMENTAL DESIGN: MLS human ovarian cancer cells were cultured in serum-free medium. Cells of passage eight were treated in combination with the anticancer agent doxorubicin at different peak plasma concentrations for 24 hours, and then maintained under suspension culture. The acquired increased aggressiveness properties was confirmed by multidrug resistance assays and by their ability to grow in an anchorage-independent manner in vitro as tumor spheroids. RESULTS: Cells selected after chemotherapy had a increased proliferative potential, eliminated Rhodamine 123 in culture and also formed spheroids in suspension. CONCLUSIONS: Here we present direct evidence that the metastasis of human ovarian cancer may be a result of transformation and dysfunction of immature precursor cells in the ovary. Also, spheroid formation may represent a key component of chemotherapy recurrence and a better understanding of these 3D structures can contribute to the development of new treatments for metastatic carcinoma.

The expression of copper transporters associated with the ototoxicity induced by platinum-based chemotherapeutic agents.

BACKGROUND: Antitumor agents based on platinum have gained a well-established place in the treatment of several forms of cancer. Their efficiency is hampered by serious toxic effects against healthy tissues as well. Ototoxicity is a serious side effect leading to hearing impairment and represents an important issue affecting the patients' quality of life. The currently used platinum chemotherapeutics exert different toxicity towards cochlear cells. The aim of our study was to answer some questions regarding the differential uptake and cellular pharmacodynamics of Cisplatin (CDDP), Carboplatin (CBDCA) and Oxaliplatin (L-OHP) in the HEI-OC1 cochlear cell line. METHODS: We studied the expression of copper transporters CTR1, ATP7A and ATP7B which are presumably involved in the uptake, cellular transport and efflux of platinum compounds by immunofluorescence microscopy and flow-cytometry. The cellular uptake of the compounds was evaluated through the determination of intracellular platinum concentration by atomic absorption spectroscopy. The effects of the treatment of HEI-OC1 cells with platinum compounds were also evaluated: cytotoxicity with the Cell Titer Blue viability test, formation of reactive oxygen species with 2',7' -dichlorofluorescein diacetate, genotoxicity with the comet assay and apoptosis with the cleaved PARP ELISA test. RESULTS: CTR1, ATP7A and ATP7B were all expressed by HEI-OC1 cells. The treatment with the platinum compounds led to a modulation of their expression, manifested in a differential platinum uptake. Treatment with Cisplatin led to the highest intracellular concentration of platinum compared to Oxaliplatin and Carboplatin at the same dose. Treatment with CuSO4 reduced platinum uptake of all the compounds, significantly in the case of Cisplatin and Carboplatin. CDDP was the most cytotoxic against HEI-OC1 cells, with an IC50 = 65.79  µM, compared to 611.7 µM for L-OHP and 882.9 µM for CBDCA, at the same molar concentration. The production of ROS was the most intense after CDDP, followed by L-OHP and CBDCA. In the comet assay, at the 100 µM concentration, L-OHP and CBDCA induced DNA adducts while CDDP induced adducts as well as DNA strand breaks. CBDCA and L-OHP lead to a significant increase of cleaved PARP at 24h (p < 0.001), suggesting an important apoptotic process induced by these compounds at the used concentrations. CONCLUSIONS: The results obtained in the current study suggest that the modulation of copper transporters locally may represent a new strategy against platinum drugs ototoxicity.

Quantification of DNA Double Strand Breaks and Oxidation Response in Children and Adults Undergoing Dental CBCT Scan.

Assessing the possible biological effects of exposure to low doses of ionizing radiation (IR) is one of the prime challenges in radiation protection, especially in medical imaging. Today, radiobiological data on cone beam CT (CBCT) related biological effects are scarce. In children and adults, the induction of DNA double strand breaks (DSBs) in buccal mucosa cells and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) and antioxidant capacity in saliva samples after CBCT examination were examined. No DNA DSBs induction was observed in children nor adults. In children only, an increase in 8-oxo-dG levels was observed 30 minutes after CBCT. At the same time an increase in antioxidant capacity was observed in children, whereas a decrease was observed in adults. Our data indicate that children and adults react differently to IR doses associated with CBCT. Fully understanding these differences could lead to an optimal use of CBCT in different age categories as well as improved radiation protection guidelines.

New insights in gene expression alteration as effect of doxorubicin drug resistance in triple negative breast cancer cells.

BACKGROUND: Triple negative breast cancer (TNBC) is a heterogeneous disease with aggressive behavior and an unfavorable prognosis rate. Due to the lack of surface receptors, TNBC must be intensely investigated in order to establish a suitable treatment for patients with this pathology. Chemoresistance is an important reason for therapeutic failure in TNBC. METHOD: The aim of this study was to investigate the effect of doxorubicin in TNBC cell lines and to highlight cellular and molecular alterations after a long exposure to doxorubicin. RESULTS: The results revealed that doxorubicin significantly increased the half maximal inhibitory concentration (IC50) values at P12 and P24 compared to parenteral cells P0. Modifications in gene expression were investigated through microarray technique, and for detection of mutational pattern was used Next Generation Sequencing (NGS). 196 upregulated and 115 downregulated genes were observed as effect of multiple dose exposure, and 15 overexpressed genes were found to be involved in drug resistance. Also, the presence of some additional mutations in both cell lines was observed. CONCLUSION: The outcomes of this research may provide novel biomarkers for drug resistance in TNBC. Also, this activity can highlight the potential mechanisms associated with drug resistance, as well as the potential therapies to counteract these mechanisms.

Effects of 60Co gamma-rays on human osteoprogenitor cells.

BACKGROUND: Radiation therapy is one of the most efficient treatments of neoplastic diseases used worldwide. However, patients who undergo radiotherapy may develop side effects that can be life threatening because tissue complications caused by radiation-induced stem cell depletion may result in structural and functional alterations of the surrounding matrix. This treatment also damages the osteogenic activity of human bone marrow by suppressing osteoblasts, leading to post-irradiation sequelae. Even if widely used in oncology, there is still little information on the fate and potential therapeutic efficacy of electromagnetic rays. MATERIAL AND METHODS: We addressed this question using both human mesenchymal stem cells and osteoblasts. Monoclonal antibody characterization identified specific surface markers for stem cells (SSEA-4, CD29, CD105, Oct 3, Nanog and SOX2) and osteoblasts (Osteopontin and Osteonectin). The technique of anti-alkaline phosphatase FITC-staining demonstrated the presence of this specific ectoenzyme. Cells were cultured in complex osteogenic medium (DMEM, 15% fetal calf serum, non-essential amino acids, L-glutamine, dexametazone, ascorbic acid, insulin, TGF-beta, BMP-2 and beta-glycero-phosphate) after being irradiated at 0.5 Gy, 1 Gy, 2 Gy and 4 Gy using a Theratron 1000 60Co source. The viability of irradiated cells was assessed using Trypan Blue staining. The comparison between cell lineages after culture in osteogenic media regarding phenotypical characterization and the intensity of the mineralization process included histology stainings (Alizarin Red S, Alcian Blue and von Kossa), and the MTT-based proliferation assay. RESULTS: After irradiation, the proliferation and differentiation of osteoprogenitor cells is dose-dependent. CONCLUSIONS: This study is one among the first papers investigating the biophysics of low-dose gamma-irradiation on stem cell culture, focusing on the potential applications in radiation oncology and various palliative treatments.

Method validation to assess in vivo cellular and subcellular changes in buccal mucosa cells and saliva following CBCT examinations.

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.

Low-dose radiations derived from cone-beam CT induce transient DNA damage and persistent inflammatory reactions in stem cells from deciduous teeth.

OBJECTIVES: Cone-beam CT (CBCT), a radiographic tool for diagnosis, treatment, and follow-up in dental practice, was introduced also in pediatric radiology, especially orthodontics. Such patients subjected to repetitive X-rays examinations may receive substantial levels of radiation doses. Ionizing radiation (IR), a recognized carcinogenic factor causing DNA double-strand breaks (DSBs) could be harmful to undifferentiated cells such as dental pulp stem cells (DPSCs) since inaccurately repaired or unrepaired DSBs may lead to malignant transformation. The H2AX and MRE11 proteins generated following DSBs formation and pro-inflammatory cytokines (CKs) secreted after irradiation are relevant candidates to monitor the cellular responses induced by CBCT. METHODS: DPSCs were extracted from human exfoliated deciduous teeth and their phenotype was assessed by immunocytochemistry and flow-cytometry. Cells were exposed to IR doses: 5.4-107.7 mGy, corresponding to 0.5-8 consecutive skull exposures, respectively. H2AX and MRE11 were detected in whole cells, while IL-1α, IL-6, IL-8, TNFα in supernatants, using enzyme-linked immunosorbent assay (ELISA) at different time points after exposure. RESULTS: The phosphorylation level of H2AX in DPSCs increased considerably at 0.5 h after exposure (p < 0.001 for 3, 5, 8 skull exposures and p < 0.05 for 1 skull exposure, respectively). MRE11 response could only be detected for the highest IR dose (p < 0.001) in the same interval. CKs secretion increased upon CBCT exposure according to doses and time. CONCLUSIONS: The DPSCs exposure to CBCT induces transient DNA damage and persistent inflammatory reaction in DPSCs drawing the attention on the potential risks of IR exposures and on the importance of dose monitoring in pediatric population.