Reduced Graphene Oxide Modulates the FAK-Dependent Signaling Pathway in Glioblastoma Multiforme Cells In Vitro.

Aggressive invasiveness is a common feature of malignant gliomas, despite their high level of tumor heterogeneity and possible diverse cell origins. Therefore, it is important to explore new therapeutic methods. In this study, we evaluated and compared the effects of graphene (GN) and reduced graphene oxides (rGOs) on a highly invasive and neoplastic cell line, U87. The surface functional groups of the GN and rGO flakes were characterized by X-ray photoelectron spectroscopy. The antitumor activity of these flakes was obtained by using the neutral red assay and their anti-migratory activity was determined using the wound healing assay. Further, we investigated the mRNA and protein expression levels of important cell adhesion molecules involved in migration and invasiveness. The rGO flakes, particularly rGO/ATS and rGO/TUD, were found highly toxic. The migration potential of both U87 and Hs5 cells decreased, especially after rGO/TUD treatment. A post-treatment decrease in mobility and FAK expression was observed in U87 cells treated with rGO/ATS and rGO/TUD flakes. The rGO/TUD treatment also reduced ß-catenin expression in U87 cells. Our results suggest that rGO flakes reduce the migration and invasiveness of U87 tumor cells and can, thus, be used as potential antitumor agents.

Equid Alphaherpesvirus 1 Modulates Actin Cytoskeleton and Inhibits Migration of Glioblastoma Multiforme Cell Line A172.

Equid alphaherpesvirus 1 (EHV-1) causes respiratory diseases, abortion, and neurological disorders in horses. Recently, the oncolytic potential of this virus and its possible use in anticancer therapy has been reported, but its influence on cytoskeleton was not evaluated yet. In the following study, we have examined disruptions in actin cytoskeleton of glioblastoma multiforme in vitro model-A172 cell line, caused by EHV-1 infection. We used three EHV-1 strains: two non-neuropathogenic (Jan-E and Rac-H) and one neuropathogenic (EHV-1 26). Immunofluorescent labelling, confocal microscopy, real-time cell growth analysis and OrisTM cell migration assay revealed disturbed migration of A172 cells infected with the EHV-1, probably due to rearrangement of actin cytoskeleton and the absence of cell projections. All tested strains caused disruption of the actin network and general depolymerization of microfilaments. The qPCR results confirmed the effective replication of EHV-1. Thus, we have demonstrated, for the first time, that EHV-1 infection leads to inhibition of proliferation and migration in A172 cells, which might be promising for new immunotherapy treatment.

Effects of Metallic and Carbon-Based Nanomaterials on Human Pancreatic Cancer Cell Lines AsPC-1 and BxPC-3.

Pancreatic cancer, due to its asymptomatic development and drug-resistance, is difficult to cure. As many metallic and carbon-based nanomaterials have shown anticancer properties, we decided to investigate their potential use as anticancer agents against human pancreatic adenocarcinoma. The objective of the study was to evaluate the toxic properties of the following nanomaterials: silver (Ag), gold (Au), platinum (Pt), graphene oxide (GO), diamond (ND), and fullerenol (C60(OH)40) against the cell lines BxPC-3, AsPC-1, HFFF-2, and HS-5. The potential cytotoxic properties were evaluated by the assessment of the cell morphology, cell viability, and cell membrane damage. The cancer cell responses to GO and ND were analysed by determination of changes in the levels of 40 different pro-inflammatory proteins. Our studies revealed that the highest cytotoxicity was obtained after the ND treatment. Moreover, BxPC-3 cells were more sensitive to ND than AsPC-1 cells due to the ND-induced ROS production. Furthermore, in both of the cancer cell lines, ND caused an increased level of IL-8 and a decreased level of TIMP-2, whereas GO caused only decreased levels of TIMP-2 and ICAM-1 proteins. This work provides important data on the toxicity of various nanoparticles against pancreatic adenocarcinoma cell lines.

Diamond Nanofilm Normalizes Proliferation and Metabolism in Liver Cancer Cells.

PURPOSE: Surgical resection of hepatocellular carcinoma can be associated with recurrence resulting from the degeneration of residual volume of the liver. The objective was to assess the possibility of using a biocompatible nanofilm, made of a colloid of diamond nanoparticles (nfND), to fill the side after tumour resection and optimize its contact with proliferating liver cells, minimizing their cancerous transformation. METHODS: HepG2 and C3A liver cancer cells and HS-5 non-cancer cells were used. An aqueous colloid of diamond nanoparticles, which covered the cell culture plate, was used to create the nanofilm. The roughness of the resulting nanofilm was measured by atomic force microscopy. Mitochondrial activity and cell proliferation were measured by XTT and BrdU assays. Cell morphology and a scratch test were used to evaluate the invasiveness of cells. Flow cytometry determined the number of cells within the cell cycle. Protein expression in was measured by mass spectrometry. RESULTS: The nfND created a surface with increased roughness and exposed oxygen groups compared with a standard plate. All cell lines were prone to settling on the nanofilm, but cancer cells formed more relaxed clusters. The surface compatibility was dependent on the cell type and decreased in the order C3A >HepG2 >HS-5. The invasion was reduced in cancer lines with the greatest effect on the C3A line, reducing proliferation and increasing the G2/M cell population. Among the proteins with altered expression, membrane and nuclear proteins dominated. CONCLUSION: In vitro studies demonstrated the antiproliferative properties of nfND against C3A liver cancer cells. At the same time, the need to personalize potential therapy was indicated due to the differential protein synthetic responses in C3A vs HepG2 cells. We documented that nfND is a source of signals capable of normalizing the expression of many intracellular proteins involved in the transformation to non-cancerous cells.

Reduced Graphene Oxides Modulate the Expression of Cell Receptors and Voltage-Dependent Ion Channel Genes of Glioblastoma Multiforme.

The development of nanotechnology based on graphene and its derivatives has aroused great scientific interest because of their unusual properties. Graphene (GN) and its derivatives, such as reduced graphene oxide (rGO), exhibit antitumor effects on glioblastoma multiforme (GBM) cells in vitro. The antitumor activity of rGO with different contents of oxygen-containing functional groups and GN was compared. Using FTIR (fourier transform infrared) analysis, the content of individual functional groups (GN/exfoliation (ExF), rGO/thermal (Term), rGO/ammonium thiosulphate (ATS), and rGO/ thiourea dioxide (TUD)) was determined. Cell membrane damage, as well as changes in the cell membrane potential, was analyzed. Additionally, the gene expression of voltage-dependent ion channels (clcn3, clcn6, cacna1b, cacna1d, nalcn, kcne4, kcnj10, and kcnb1) and extracellular receptors was determined. A reduction in the potential of the U87 glioma cell membrane was observed after treatment with rGO/ATS and rGO/TUD flakes. Moreover, it was also demonstrated that major changes in the expression of voltage-dependent ion channel genes were observed in clcn3, nalcn, and kcne4 after treatment with rGO/ATS and rGO/TUD flakes. Furthermore, the GN/ExF, rGO/ATS, and rGO/TUD flakes significantly reduced the expression of extracellular receptors (uPar, CD105) in U87 glioblastoma cells. In conclusion, the cytotoxic mechanism of rGO flakes may depend on the presence and types of oxygen-containing functional groups, which are more abundant in rGO compared to GN.

Use of Selected Carbon Nanoparticles as Melittin Carriers for MCF-7 and MDA-MB-231 Human Breast Cancer Cells.

Despite advanced techniques in medicine, breast cancer caused the deaths of 627,000 women in 2018. Melittin, the main component of bee venom, has lytic properties for many types of cells, including cancer cells. To increase its toxic effect, carbon nanoparticles, graphene oxide, pristine graphene, and diamond were used as carriers of melittin to breast cancer cells. To date, the effects of carbon nanoparticles as carriers of melittin on cancer cells have not been studied. The present study was carried out on MCF-7 and MDA-MB-231 cell lines. The investigation consisted of structural analysis of complexes using transmission electron microscopy, zeta potential measurements, evaluation of cell morphology, assessment of cell viability and membrane integrity, investigation of reactive oxygen species production, and investigation of mitochondrial membrane potential. Cell death was examined by flow cytometry and a membrane test for 43 apoptotic proteins. The results indicate that melittin complex with nanographene oxide has a stronger toxic effect on breast cancer cells than melittin alone. Moreover, nanodiamonds can protect cells against the lytic effects of melittin. All complexes reduced, but not completely eliminated the level of necrosis, compared to melittin. Thus, results suggest that the use of carbon nanoparticles as carriers for melittin may find use in medicine in the future.

Influence of Selected Carbon Nanostructures on the CYP2C9 Enzyme of the P450 Cytochrome.

Carbon nanostructures have recently gained significant interest from scientists due to their unique physicochemical properties and low toxicity. They can accumulate in the liver, which is the main expression site of cytochrome P450 (CYP450) enzymes. These enzymes play an important role in the metabolism of exogenous compounds, such as drugs and xenobiotics. Altered activity or expression of CYP450 enzymes may lead to adverse drug effects and toxicity. The objective of this study was to evaluate the influence of three carbon nanostructures on the activity and expression at the mRNA and protein levels of CYP2C9 isoenzyme from the CYP2C subfamily: Diamond nanoparticles, graphite nanoparticles, and graphene oxide platelets. The experiments were conducted using two in vitro models. A microsome model was used to assess the influence of the three-carbon nanostructures on the activity of the CYP2C9 isoenzyme. The CYP2C9 gene expression at the mRNA and protein levels was determined using a hepatoma-derived cell line HepG2. The experiments have shown that all examined nanostructures inhibit the enzymatic activity of the studied isoenzymes. Moreover, a decrease in the expression at the mRNA and protein levels was also observed. This indicates that despite low toxicity, the nanostructures can alter the enzymatic function of CYP450 enzymes, and the molecular pathways involved in their expression.

Mechano-signalling, induced by fullerene C60 nanofilms, arrests the cell cycle in the G2/M phase and decreases proliferation of liver cancer cells.

INTRODUCTION AND OBJECTIVE: Degradation of the extracellular matrix (ECM) changes the physicochemical properties and dysregulates ECM-cell interactions, leading to several pathological conditions, such as invasive cancer. Carbon nanofilm, as a biocompatible and easy to functionalize material, could be used to mimic ECM structures, changing cancer cell behavior to perform like normal cells. METHODS: Experiments were performed in vitro with HS-5 cells (as a control) and HepG2 and C3A cancer cells. An aqueous solution of fullerene C60 was used to form a nanofilm. The morphological properties of cells cultivated on C60 nanofilms were evaluated with light, confocal, electron and atomic force microscopy. The cell viability and proliferation were measured by XTT and BrdU assays. Immunoblotting and flow cytometry were used to evaluate the expression level of proliferating cell nuclear antigen and determine the number of cells in the G2/M phase. RESULTS: All cell lines were spread on C60 nanofilms, showing a high affinity to the nanofilm surface. We found that C60 nanofilm mimicked the niche/ECM of cells, was biocompatible and non-toxic, but the mechanical signal from C60 nanofilm created an environment that affected the cell cycle and reduced cell proliferation. CONCLUSION: The results indicate that C60 nanofilms might be a suitable, substitute component for the niche of cancer cells. The incorporation of fullerene C60 in the ECM/niche may be an alternative treatment for hepatocellular carcinoma.

Effect of Juice and Extracts from Saposhnikovia divaricata Root on the Colon Cancer Cells Caco-2.

Colorectal cancer ranks 3rd in terms of cancer incidence. Growth and development of colon cancer cells may be affected by juice and extracts from Saposhnikovia divaricata root. The objective of the research was to analyze the effect of S. divaricata juice and extracts on the viability, membrane integrity and types of cell death of Caco-2 cells. Juice and extracts were analyzed using Ultra-High Performance Liquid Chromatography-Mass Spectrometry (UHPLC-MS) and in respect of the presence of antioxidants, total carbohydrates, protein, fat and polyphenols. The contents of cimifugin ß-D-glucopyranoside, cimifugin, 4'-O-glucopyranosyl-5-O-methylvisamminol, imperatorin and protein were the highest in juice. 50% Hydroethanolic extract had the greatest antioxidant potential, concentration of polyphenols and fat. Water extract was characterized by the highest content of glutathione. Juice and 75% hydroethanolic extract contained the most carbohydrates. After the application of juice, 50% extract and the juice fraction containing the molecules with molecular weights >50 kDa, a decrease of the cell viability was noted. Juice and this extract exhibited the protective properties in relation to the cell membranes and they induced apoptosis. The knowledge of further mechanisms of anticancer activity of the examined products will allow to consider their use as part of combination therapy.

Diamond Nanoparticles Downregulate Expression of CycD and CycE in Glioma Cells.

Our previous studies have shown that diamond nanoparticles (NDs) exhibited antiangiogenic and proapoptotic properties in vitro in glioblastoma multiforme (GBM) cells and in tumors in vivo. Moreover, NDs inhibited adhesion, leading to the suppression of migration and invasion of GBM. In the present study, we hypothesized that the NDs might also inhibit proliferation and cell cycle in glioma cells. Experiments were performed in vitro with the U87 and U118 lines of GBM cells, and for comparison, the Hs5 line of stromal cells (normal cells) after 24 h and 72 h of treatment. The analyses included cell morphology, cell death, viability, and cell cycle analysis, double timing assay, and gene expression (Rb, E2F1, CycA, CycB, CycD, CycE, PTEN, Ki-67). After 72 h of ND treatment, the expression level of Rb, CycD, and CycE in the U118 cells, and E2F1, CycD, and CycE in the U87 cells were significantly lower in comparison to those in the control group. We observed that decreased expression of cyclins inhibited the G1/S phase transition, arresting the cell cycle in the G0/G1 phase in glioma cells. The NDs did not affect the cell cycle as well as PTEN and Ki-67 expression in normal cells (Hs5), although it can be assumed that the NDs reduced proliferation and altered the cell cycle in fast dividing cells.

Evaluation of in vitro effect of selected contact lens solutions conjugated with nanoparticles in terms of preventive approach to public health risk generated by Acanthamoeba strains.

INTRODUCTION: Various Acanthamoeba species are free-living organisms widely distributed in the human environment. Amphizoic amoebae as facultative parasites may cause vision-threatening eye disease - Acanthamoeba keratitis, mostly among contact lens wearers. As the number of cases is increasing, and applied therapy often unsuccessful, proper hygienic measures and effective contact lenses disinfection are crucial for the prevention of this disease. Available contact lens solutions are not fully effective against amphizoic amoebae; there is a need to enhance their disinfecting activity to prevent amoebic infections. The use of developing nanotechnology methods already applied with success in the prevention, diagnostic and therapy of other infectious diseases might be helpful regarding amoebic keratitis. This study assesses the in vitro effect of selected contact lens solutions conjugated with nanoparticles against Acanthamoeba trophozoites. MATERIAL AND METHODS: Three selected contact lens solutions conjugated with silver and gold nanoparticles in concentration of 0.25-2.5 ppm were used in vitro against the axenically cultured ATCC 30010 type Acanthamoeba castellanii strain. The anti-amoebic efficacy was examined based on the oxido-reduction of AlamarBlue. The cytotoxicity tests based on the measurement of lactate dehydrogenase (LDH) activity were performed using a fibroblast HS-5 cell line. RESULTS: Enhancement of the anti-amoebic activity of contact lens solutions conjugated with selected nanoparticles expressed in the dose dependent amoebic growth inhibition with a low cytotoxicity profile was observed. CONCLUSIONS: Results of the study showed that conjugation of selected contact lens solutions with silver nanoparticles might be a promising approach to prevent Acanthamoeba keratitis among contact lens users.

Nanostructures of diamond, graphene oxide and graphite inhibit CYP1A2, CYP2D6 and CYP3A4 enzymes and downregulate their genes in liver cells.

INTRODUCTION AND OBJECTIVE: Currently, carbon nanostructures are vastly explored materials with potential for future employment in biomedicine. The possibility of employment of diamond nanoparticles (DN), graphene oxide (GO) or graphite nanoparticles (GN) for in vivo applications raises a question of their safety. Even though they do not induce a direct toxic effect, due to their unique properties, they can still interact with molecular pathways. The objective of this study was to assess if DN, GO and GN affect three isoforms of cytochrome P450 (CYP) enzymes, namely, CYP1A2, CYP2D6 and CYP3A4, expressed in the liver. METHODS: Dose-dependent effect of the DN, GO and GN nanostructures on the catalytic activity of CYPs was examined using microsome-based model. Cytotoxicity of DN, GO and GN, as well as the influence of the nanostructures on mRNA expression of CYP genes and CYP-associated receptor genes were studied in vitro using HepG2 and HepaRG cell lines. RESULTS: All three nanostructures interacted with the CYP enzymes and inhibited their catalytic activity in microsomal-based models. CYP gene expression at the mRNA level was also downregulated in HepG2 and HepaRG cell lines. Among the three nanostructures, GO showed the most significant influence on the enzymes, while DN was the most inert. CONCLUSION: Our findings revealed that DN, GO and GN might interfere with xenobiotic and drug metabolism in the liver by interactions with CYP isoenzymes responsible for the process. Such results should be considered if DN, GO and GN are used in medical applications.

Effects of Reduced Graphene Oxides on Apoptosis and Cell Cycle of Glioblastoma Multiforme.

Graphene (GN) and its derivatives (rGOs) show anticancer properties in glioblastoma multiforme (GBM) cells in vitro and in tumors in vivo. We compared the anti-tumor effects of rGOs with different oxygen contents with those of GN, and determined the characteristics of rGOs useful in anti-glioblastoma therapy using the U87 glioblastoma line. GN/ExF, rGO/Term, rGO/ATS, and rGO/TUD were structurally analysed via transmission electron microscopy, Raman spectroscopy, FTIR, and AFM. Zeta potential, oxygen content, and electrical resistance were determined. We analyzed the viability, metabolic activity, apoptosis, mitochondrial membrane potential, and cell cycle. Caspase- and mitochondrial-dependent apoptotic pathways were investigated by analyzing gene expression. rGO/TUD induced the greatest decrease in the metabolic activity of U87 cells. rGO/Term induced the highest level of apoptosis compared with that induced by GN/ExF. rGO/ATS induced a greater decrease in mitochondrial membrane potential than GN/ExF. No significant changes were observed in the cytometric study of the cell cycle. The effectiveness of these graphene derivatives was related to the presence of oxygen-containing functional groups and electron clouds. Their cytotoxicity mechanism may involve electron clouds, which are smaller in rGOs, decreasing their cytotoxic effect. Overall, cytotoxic activity involved depolarization of the mitochondrial membrane potential and the induction of apoptosis in U87 glioblastoma cells.