Biological Effects of Small Sized Graphene Oxide Nanosheets on Human Leukocytes.

Since the discovery of graphene, there has been a wide range of the literature dealing with its versatile structure and easy binding of biomolecules as well as its large loading capacity. In the emerging field of immunotherapy, graphene and its derivatives have potential uses as drug delivery platforms directly into tumour sites or as adjuvants in cancer vaccines, as they are internalized by monocytes which in turn may activate adaptive anti-tumoral immune responses. In this study, we expose cells of the innate immune system and a human acute monocytic leukemia cell line (THP-1) to low doses of small-sized GO nanosheets functionalized with bovine serum albumin (BSA) and fluorescein isothiocyanate (FITC), to study their acute response after internalization. We show by flow cytometry, uptake in cells of GO-BSA-FITC reaches 80% and cell viability and ROS production are both unaffected by exposure to nanoparticles. On the contrary, GO-BSA nanosheets seem to have an inhibitory effect on ROS production, probably due to their antioxidant properties. We also provided results on chemotaxis of macrophages derived from peripheral blood monocytes treated with GO-BSA. In conclusion, we showed the size of nanosheets, the concentration used and the degree of functionalization were important factors for biocompatibility of GO in immune cells. Its low cytotoxicity and high adaptability to the cells of the innate immune system make it a good candidate for deployment in immunotherapy, in particular for delivering protein antigens to monocytes which activate adaptive immunity.

HHV-6A Infection of Papillary Thyroid Cancer Cells Induces Several Effects Related to Cancer Progression.

Recent studies have shown that thyrocytes are permissive to HHV-6A infection and that the virus may contribute to the pathogenesis of autoimmune thyroiditis. Thyroid autoimmune diseases increase the risk of papillary cancer, which is not surprising considering that chronic inflammation activates pathways that are also pro-oncogenic. Moreover, in this condition, cell proliferation is stimulated as an attempt to repair tissue damage caused by the inflammatory process. Interestingly, it has been reported that the well-differentiated papillary thyroid carcinoma (PTC), the less aggressive form of thyroid tumor, may progress to the more aggressive follicular thyroid carcinoma (FTC) and eventually to the anaplastic thyroid carcinoma (ATC), and that to such progression contributes the presence of an inflammatory/immune suppressive tumor microenvironment. In this study, we investigated whether papillary tumor cells (BCPAP) could be infected by human herpes virus-6A (HHV-6A), and if viral infection could induce effects related to cancer progression. We found that the virus dysregulated the expression of several microRNAs, such as miR-155, miR-9, and the miR-221/222 cluster, which are involved in different steps of carcinogenesis, and increased the secretion of pro-inflammatory cytokines, particularly IL-6, which may also sustain thyroid tumor cell growth and promote cancer progression. Genomic instability and the expression of PTEN, reported to act as an oncogene in mutp53-carrying cells such as BCPAP, also increased following HHV-6A-infection. These findings suggest that a ubiquitous herpesvirus such as HHV-6A, which displays a marked tropism for thyrocytes, could be involved in the progression of PTC towards more aggressive forms of thyroid tumor.

High mobility group box 1 in women with unexplained recurrent pregnancy loss.

OBJECTIVES: To investigate whether high mobility group box 1 (HMGB1) is involved in unexplained recurrent pregnancy loss (uRPL). METHODS: Plasma levels of HMGB1 were measured by ELISA in non-pregnant women with (n=44) and without (n=53 controls) uRPL. Their platelets and plasma-derived microvesicles (MVs) were also assayed for HMGB1. Endometrial biopsies were taken in selected uRPL (n=5) and control women (n=5) and the tissue expression of HMGB1 was determined by western blot and immunohistochemistry (IHC). RESULTS: plasma levels of HMGB1 were significantly higher in women with uRPL than in control women. HMGB1 content in platelets and MVs obtained from women with uRPL was significantly higher than that obtained from control women. HMGB1 expression in endometrium was higher in tissues obtained from women with uRPL than in tissues obtained from control women. IHC analysis revealed that HMGB1 is expressed in endometrium with different patterns between uRPL and control women. CONCLUSIONS: HMGB1 could be involved in uRPL.

ZnO Nanorods Create a Hypoxic State with Induction of HIF-1 and EPAS1, Autophagy, and Mitophagy in Cancer and Non-Cancer Cells.

Nanomaterials are gaining increasing attention as innovative materials in medicine. Among nanomaterials, zinc oxide (ZnO) nanostructures are particularly appealing because of their opto-electrical, antimicrobial, and photochemical properties. Although ZnO is recognized as a safe material and the Zn ion (Zn2+) concentration is strictly regulated at a cellular and systemic level, different studies have demonstrated cellular toxicity of ZnO nanoparticles (ZnO-NPs) and ZnO nanorods (ZnO-NRs). Recently, ZnO-NP toxicity has been shown to depend on the intracellular accumulation of ROS, activation of autophagy and mitophagy, as well as stabilization and accumulation of hypoxia-inducible factor-1α (HIF-1α) protein. However, if the same pathway is also activated by ZnO-NRs and how non-cancer cells respond to ZnO-NR treatment, are still unknown. To answer to these questions, we treated epithelial HaCaT and breast cancer MCF-7 cells with different ZnO-NR concentrations. Our results showed that ZnO-NR treatments increased cell death through ROS accumulation, HIF-1α and endothelial PAS domain protein 1 (EPAS1) activation, and induction of autophagy and mitophagy in both cell lines. These results, while on one side, confirmed that ZnO-NRs can be used to reduce cancer growth, on the other side, raised some concerns on the activation of a hypoxic response in normal cells that, in the long run, could induce cellular transformation.

Extra-Cellular Vesicles Derived from Thyroid Cancer Cells Promote the Epithelial to Mesenchymal Transition (EMT) and the Transfer of Malignant Phenotypes through Immune Mediated Mechanisms.

Thyroid cancer is the most common endocrine cancer, and its incidence is increasing in many countries around the world. Among thyroid cancers, the papillary thyroid cancer (PTC) histotype is particularly prevalent. A small percentage of papillary tumors is associated with metastases and aggressive behavior due to de-differentiation obtained through the epithelial-mesenchymal transition (EMT) by which epithelial thyroid cells acquire a fibroblast-like morphology, reduce cellular adhesion, increase motility and expression of mesenchymal proteins. The tumor microenvironment plays an important role in promoting an aggressive phenotype through hypoxia and the secretion of HMGB1 and other factors. Hypoxia has been shown to drastically change the tumor cell phenotype and has been associated with increasing metastatic and migratory behavior. Cells transfer information to neighboring cells or distant locations by releasing extracellular membrane vesicles (EVs) that contain key molecules, such as mRNAs, microRNAs (miRNAs), and proteins, that are able to modify protein expression in recipient cells. In this study, we investigated the potential role of EVs released by the anaplastic cancer cell line CAL-62 in inducing a malignant phenotype in a papillary cancer cell line (BCPAP).

GO Nanosheets: Promising Nano Carrier for the S29, 1-(2-Chloro-2-(4-chlorophenyl-ethyl)-N-(4-fluorobenzyl)-1H-pyrazolo[3,4-d] pyrimidin-4-amine, Therapeutic Agent in Neuroblastoma.

Graphene oxide (GO) derivatives are reported as a valid alternative to conventional carriers of therapeutic agents, because they have a large surface area, an excellent electrical and thermal conductivity and a great capacity for selective binding of drugs and therapeutics, due to the functionalization of their surfaces, edges and sides. In this work GO nanosheets, synthesized by electrochemical exfoliation of graphite (patent N 102015000023739, Tor Vergata University), were investigated as possible carriers of an anticancer drug, the S29, an inhibitor of a cytoplasmic tyrosine kinase (c-SRC) on a neuroblastoma cell line (SK N BE 2 cells). Neuroblastoma is a heterogenous tumor whose characteristics range from spontaneous regression to aggressive phenotypes that are due to different mutations that often occur in SRC family kinases. Inhibitors of tyrosine kinases are currently investigated for their anti-tumoral effects on aggressive neuroblastomas, but their uptake in cells and pharmacokinetics needs to be improved. In this work S29 was stably conjugated with highly water-dispersible GO nanoparticles. S29/GO complex formation was induced by 1h sonication and its stability was analyzed by chromatography coupled with spectrophotometry and mass spectrometry. The synthesized composite (GO-S29) was delivered into SK N BE 2 cells and its effects on cell viability, production of reactive oxygen species (ROS) and migration were studied. The results show that the compound GO-S29 exerts anti-tumoral effects on the neuroblastoma cell line, higher than both GO and S29 do alone and that GO has an additive effect on S29.

Non-Coding RNA: Role in Gestational Diabetes Pathophysiology and Complications.

Gestational Diabetes Mellitus (GDM) is defined as glucose intolerance that develops in the second or third trimester of pregnancy. GDM can lead to short-term and long-term complications both in the mother and in the offspring. Diagnosing and treating this condition is therefore of great importance to avoid poor pregnancy outcomes. There is increasing interest in finding new markers with potential diagnostic, prognostic and therapeutic utility in GDM. Non-coding RNAs (ncRNAs), including microRNAs, long non-coding RNAs and circular RNAs, are critically involved in metabolic processes and their dysregulated expression has been reported in several pathological contexts. The aberrant expression of several circulating or placenta-related ncRNAs has been linked to insulin resistance and ß-cell dysfunction, the key pathophysiological features of GDM. Furthermore, significant associations between altered ncRNA profiles and GDM-related complications, such as macrosomia or trophoblast dysfunction, have been observed. Remarkably, the deregulation of ncRNAs, which might be linked to a detrimental intrauterine environment, can lead to changes in the expression of target genes in the offspring, possibly contributing to the development of long-term GDM-related complications, such as metabolic and cardiovascular diseases. In this review, all the recent findings on ncRNAs and GDM are summarized, particularly focusing on the molecular aspects and the pathophysiological implications of this complex relationship.

Artichoke Polyphenols Sensitize Human Breast Cancer Cells to Chemotherapeutic Drugs via a ROS-Mediated Downregulation of Flap Endonuclease 1.

Combined treatment of several natural polyphenols and chemotherapeutic agents is more effective comparing to the drug alone in inhibiting cancer cell growth. Polyphenolic artichoke extracts (AEs) have been shown to have anticancer properties by triggering apoptosis or reactive oxygen species- (ROS-) mediated senescence when used at high or low doses, respectively. Our aim was to explore the chemosensitizing potential of AEs in order to enhance the efficacy of conventional chemotherapy in breast cancer cells. We employed breast cancer cell lines to assess the potential synergistic effect of a combined treatment of AEs/paclitaxel (PTX) or AEs/adriamycin (ADR) and to determine the underlying mechanisms correlated to this potential therapeutic approach. Our data shows that AEs/PTX reduced cell proliferation by increasing DNA damage response (DDR) mediated by Flap endonuclease 1 (FEN1) downregulation that results into enhanced breast cancer cell sensitivity to chemotherapeutic drugs. We demonstrated that ROS/Nrf2 and p-ERK pathways are two molecular mechanisms involved in the synergistic effect of AEs plus PTX treatment. To highlight the role of ROS herein, we report that the addition of antioxidant N-acetylcysteine (NAC) significantly decreased the antiproliferative effect of the combined treatment. A combined therapy could be able to reduce the dose of chemotherapeutic drugs, minimizing toxicity and side effects. Our results suggest the use of artichoke polyphenols as ROS-mediated sensitizers of chemotherapy paving the way for innovative and promising natural compound-based therapeutic strategies in oncology.

Cancer immunotherapy through the prism of adaptation: Will Achilles catch the tortoise?

There is no secret that despite the rapid development of new methods of cancer therapy, we still are not able to completely destroy the tumor. Every time we attack the tumor, the tumor neutralizes our attempts. Carcinogenesis can be presented as a tree whose branches are different pro-tumor mechanisms and whose trunk is a biological phenomenon that "feeds" those branches. A tree can be destroyed in two ways: either by cutting a branch for a branch without a guarantee that new branches will not grow, or cutting down the trunk and letting the branches wither away. To cut down the trunk, it is necessary to understand the nature of the biological phenomenon, which helps the tumor to avoid attack by the immune system, drugs and immunotherapy. The clue is that the pro-tumor mechanisms are united by one goal - to increase the resistance of the tumor cell to immune factors and drugs. A phenomenon that improves cell resistance is well known in biology - adaptation. If the immunity does not immediately destroy the tumor cell, the cell begins to adapt to it. Our hypothesis is that short range adaptation to immune factors plays a role in the formation of tumor tolerance for immunity and immunotherapy. This gives rise to the idea of reducing the survival of tumor cells by disrupting adaptation mechanisms. Indeed, "turning off" the immune system for a period of time before therapy and applying immunotherapy only to tumor cells that have lost their increased resistance could be a new approach to increase the effectiveness of immunotherapy.

Metal Free Graphene Oxide (GO) Nanosheets and Pristine-Single Wall Carbon Nanotubes (p-SWCNTs) Biocompatibility Investigation: A Comparative Study in Different Human Cell Lines.

The in vitro biocompatibility of Graphene Oxide (GO) nanosheets, which were obtained by the electrochemical exfoliation of graphite electrodes in an electrolytic bath containing salts, was compared with the pristine Single Wall Carbon Nanotubes (p-SWCNTs) under the same experimental conditions in different human cell lines. The cells were treated with different concentrations of GO and SWCNTs for up to 48 h. GO did not induce any significant morphological or functional modifications (demonstrating a high biocompatibility), while SWNCTs were toxic at any concentration used after a few hours of treatment. The cell viability or cytotoxicity were detected by the trypan blue assay and the lactate dehydrogenase LDH quantitative enzymatic test. The Confocal Laser Scanning Microscopy (CLSM) and transmission electron microscopy (TEM) analysis demonstrated the uptake and internalization of GO sheets into cells, which was localized mainly in the cytoplasm. Different results were observed in the same cell lines treated with p-SWCNTs. TEM and CLSM (Confocal Laser Scanning Microscopy) showed that the p-SWCNTs induced vacuolization in the cytoplasm, disruption of cellular architecture and damage to the nuclei. The most important result of this study is our finding of a higher GO biocompatibility compared to the p-SWCNTs in the same cell lines. This means that GO nanosheets, which are obtained by the electrochemical exfoliation of a graphite-based electrode (carried out in saline solutions or other physiological working media) could represent an eligible nanocarrier for drug delivery, gene transfection and molecular cell imaging tests.

From Human Megakaryocytes to Platelets: Effects of Aspirin on High-Mobility Group Box 1/Receptor for Advanced Glycation End Products Axis.

Platelets (PLTs) are the major source of high-mobility group box 1 (HMGB1), a protein that is involved in sterile inflammation of blood vessels and thrombosis. Megakaryocytes (MKs) synthesize HMGB1 and transfer both protein and mRNA into PLTs and PLT-derived microvesicles (MV). Free HMGB1 found in supernatants of in vitro differentiated MKs and in a megakaryoblastic cell line (DAMI cells). Aspirin "in vivo" and "in vitro" not only reduces HMGB1 and receptor for advanced glycation end products expression on MKs and PLTs but also drives the movement of HMGB1 from MKs into PLTs and PLT-derived MV. These findings suggest that consumption of low doses of aspirin reduces the risk of atherosclerosis complications as well as reducing PLT aggregation by the inhibition of COX-1.

Graphene Oxide Nanoribbons Induce Autophagic Vacuoles in Neuroblastoma Cell Lines.

Since graphene nanoparticles are attracting increasing interest in relation to medical applications, it is important to understand their potential effects on humans. In the present study, we prepared graphene oxide (GO) nanoribbons by oxidative unzipping of single-wall carbon nanotubes (SWCNTs) and analyzed their toxicity in two human neuroblastoma cell lines. Neuroblastoma is the most common solid neoplasia in children. The hallmark of these tumors is the high number of different clinical variables, ranging from highly metastatic, rapid progression and resistance to therapy to spontaneous regression or change into benign ganglioneuromas. Patients with neuroblastoma are grouped into different risk groups that are characterized by different prognosis and different clinical behavior. Relapse and mortality in high risk patients is very high in spite of new advances in chemotherapy. Cell lines, obtained from neuroblastomas have different genotypic and phenotypic features. The cell lines SK-N-BE(2) and SH-SY5Y have different genetic mutations and tumorigenicity. Cells were exposed to low doses of GO for different times in order to investigate whether GO was a good vehicle for biological molecules delivering individualized therapy. Cytotoxicity in both cell lines was studied by measuring cellular oxidative stress (ROS), mitochondria membrane potential, expression of lysosomial proteins and cell growth. GO uptake and cytoplasmic distribution of particles were studied by Transmission Electron Microscopy (TEM) for up to 72 h. The results show that GO at low concentrations increased ROS production and induced autophagy in both neuroblastoma cell lines within a few hours of exposure, events that, however, are not followed by growth arrest or death. For this reason, we suggest that the GO nanoparticle can be used for therapeutic delivery to the brain tissue with minimal effects on healthy cells.

Action of HMGB1 on miR-221/222 cluster in neuroblastoma cell lines.

microRNA (miR/miRNA) are small non-coding RNAs that control gene expression at the post-transcriptional level by targeting mRNAs. Aberrant expression of miRNAs is often observed in different types of cancer. Specific miRNAs function as tumor suppressors or oncogenes and interfere with various aspects of carcinogenesis, including differentiation, proliferation and invasion. Upregulation of miRNAs 221 and 222 has been shown to induce a malignant phenotype in numerous human cancers via inhibition of phosphatase and tensin homolog (PTEN) expression. Neuroblastoma is the most common extracranial solid malignancy in children, which is characterized by cellular heterogeneity that corresponds to different clinical outcomes. The different cellular phenotypes are associated with different gene mutations and miRs that control genetic and epigenetic factors. For this reason miRs are considered a potential therapeutic target in neuroblastoma. The aim of the present study was to investigate the mechanisms by which extracellular high mobility group box 1 (HMGB1) promotes cell growth in neuroblastoma. SK-N-BE(2) and SH-SY5Y neuroblastoma derived cell lines were transfected with the antisense oligonucleotides, anti-miR-221 and -222, followed by treatment with HMGB1 to investigate the expression of the oncosuppressor PTEN. In this study, it was demonstrated that HMGB1, which is released by damaged cells and tumor cells, upregulates miR-221/222 oncogenic clusters in the two human neuroblastoma derived cell lines. The results revealed that the oncogenic cluster miRs 221/222 were more highly expressed by the most undifferentiated cell line [SK-N-BE(2)] compared with the the less tumorigenic cell line (SH-SY5Y) and that exogenous HMGB1 increases this expression. In addition, HMGB1 modulates PTEN expression via miR-221/222, as demonstrated by transiently blocking miR-221/222 with anti-sense oligonucleotides. These results may lead to the development of novel therapeutic strategies for neuroblastoma.

HMGB1 induces the overexpression of miR-222 and miR-221 and increases growth and motility in papillary thyroid cancer cells.

Experimental and epidemiological studies have revealed that chronic inflammation contributes to cancer progression and even predisposes to cellular transformation. Inflammatory infiltrates in papillary thyroid cancer include lymphocytes, macrophages and cytokines. High-mobility group box 1 protein (HMGB1) is a late inflammatory cytokine that signals danger to the immune system through the receptor for advanced glycation end-products (RAGE) and Toll-like receptor. The activation of the above receptors results in the secretion of growth, chemotactic and angiogenic factors that contribute to chronic inflammation. In this study, we suggest that apart from the activation of signal transduction pathways by the activation of RAGE, the indirect inhibition of cell cycle regulators [such as phosphatase and tensin homolog (PTEN)] may also cause an increase in cell growth and motility. MicroRNAs (miRNAs) have increasingly been implicated in regulating the malignant progression of cancer. MiR-221 and miR-222 have been found to be deregulated in human papillary thyroid carcinomas. They are involved in cell proliferation through the inhibition of the cell cycle regulator, p27kip1, in human papillary carcinomas. In this study, we show that HMGB1 increases the expression of miR-221 and miR-222 in primary cultures of excised papillary lesions and in an established papillary cancer cell line (BC PAP). The overexpression of oncogenic miR-221 and miR-222 caused by HMGB1 is associated with an increase in malignancy scores, namely cell growth and motility.

Cross-talk between NO and HMGB1 in lymphocytic thyroiditis and papillary thyroid cancer.

The controversy on whether or not inflammatory infiltrates in chronic lymphocytic thyroiditis predispose to cancer, has now merged into a debate over the role of the inflammatory infiltrates. The question is how and why some cells become transformed and what factors allow them to spread and in some cases become invasive. Here, we show that the amount of inflammatory mediators such as nitric oxide (NO) and high mobility group Box 1 protein (HMGB1) produced in thyroiditis microenvironment increases in tumors and could be involved in the cellular transformation process. NO and HMGB1 are known to attract macrophages that would promote angiogenesis, matrix remodelling and suppression of an efficient immune response. Inflammatory infiltrates could increase the risk of papillary cancer in patients with autoimmune lymphocytic thyroiditis. Cytokines and soluble inflammatory mediators involved in cancer-related inflammation are not only a target for innovative diagnostic and therapeutic strategies but they also represent a future challenge for oncologists.

Clinical and biological relationship between chronic lymphocytic thyroiditis and papillary thyroid carcinoma.

The association between chronic lymphocytic thyroiditis and papillary thyroid carcinoma has been investigated for several years from different perspectives but with few attempts to design a common frame of reference to understand the complex mutual interactions between the various pathways of inflammatory response and of thyroid tumor induction and progression. This article reviews the current knowledge and research on this topic according to epidemiologic, immunobiologic, pathologic, and biomolecular points of view, highlighting achievements and lack of knowledge. It draws some conclusions and points at possible future directions for research.

Survivin and cyclin D1 are jointly expressed in thyroid papillary carcinoma and microcarcinoma.

In order to define more effective predictive markers for clinical management and prognosis, we evaluated the expression of cyclin D1 and survivin in large papillary thyroid carcinoma (LPTC) and microcarcinoma (PTM). Sixty-seven patients operated for papillary carcinoma (36 of which with PTM) were considered. Immunochemistry for cyclin D1 and survivin was performed in samples from tumor mass and nodal metastases. There were not significant differences between LPTC and PTM as to patients personal data, TNM or MACIS staging, nodal invasion and multifocality, while capsular invasion was significantly more frequent in LPTC. Cyclin D1 and survivin were expressed at a very high rate and almost to the same extent in LPTC and PTM, both in tumoral mass and in nodal metastases. Survivin showed only cytoplasmic expression. Cyclin D1 and survivin over-expression are probably early events in tumorigenesis of thyroid papillary carcinoma but their full role in the process of tumor progression and their clinical value are still to be investigated.

Phenotypic and functional characterization of lymphocytes in autoimmune thyroiditis and in papillary carcinoma.

BACKGROUND: Infiltrates of lymphocytes are found in both autoimmune thyroid disease and papillary cancer and are responsible for thyroid destruction in autoimmune disease. Their role in neoplastic transformation is not yet clear. MATERIALS AND METHODS: Phenotypic studies and the capacity to undergo apoptosis were assessed on peripheral and gland infiltrating lymphocytes from patients with autoimmune thyroiditis and papillary carcinoma. RESULTS: Peripheral lymphocytes in these patients belong to the same phenotype as the infiltrating lymphocytes. A mixed immune response Tc2 and Tc1 is present in thyroid glands of patients with papillary tumors and the capacity to undergo apoptosis in peripheral lymphocytes from both groups of patients increases. CONCLUSION: We suggest that a switch from a Th1 (Tc1) in autoimmune thyroid disease to a Th2 or mixed response in papillary carcinoma patients in peripheral blood may help the early diagnosis of thyroid cancer and could be used in autoimmune thyroid disease patient follow-up.