miRNA patterns in male LUSC patients - the 3-way mirror: Tissue, plasma and exosomes.

Lung cancer remains one of the leading causes of cancer-related deaths worldwide. It is classified into two main histological groups: non-small cell lung cancer (NSCLC) and small cell lung cancer. Improving the outcome of cancer patients could be possible by enhancing the early diagnosis. In the current study, we evaluated the levels of three microRNAs - miR-21-5p, miR-155-5p, and miR-181a-5p in tumor (TT) vs adjacent normal tissue (NT), as well as their expression levels in plasma and extracellular vesicles (EVs) from plasma in lung squamous cell carcinoma (LUSC) male patients vs healthy individuals as means to identify a panel of miRNAs that could serve as novel biomarkers for the diagnosis of LUSC in male patients. Matched paired tissue samples from male LUSC (n=40) patients were used for miRNA expression analysis. MiR-21-5p and miR-155-5p in tumor tissue were overexpressed, while underexpression of miR-181a-5p was observed in LUSC TT vs NT. These results were further validated in the TCGA LUSC dataset, considering 279 male samples. These alterations of miR-21-5p, miR-181a-5p, and miR-155-5p in tumor tissue are also present in plasma and plasma extracellular vesicles in LUSC male patients. In addition, ROC curves were performed to assess the sensitivity and specificity of different combinations of these miRNAs, confirming a high diagnostic accuracy for LUSC of up to 88 % in male subjects. The expression levels in tissue samples and the abundance in plasma and plasma EVs of the three miRNAs combined - miR-21-5p, miR-155-5p and miR-181a-5p - could be considered for further studies on biomarkers for the early detection of LUSC in male subjects.

Current trends in luminescence-based assessment of apoptosis.

Apoptosis, the most extensively studied type of cell death, is known to play a crucial role in numerous processes such as elimination of unwanted cells or cellular debris, growth, control of the immune system, and prevention of malignancies. Defective regulation of apoptosis can trigger various diseases and disorders including cancer, neurological conditions, autoimmune diseases and developmental disorders. Knowing the nuances of the cell death type induced by a compound can help decipher which therapy is more effective for specific diseases. The detection of apoptotic cells using classic methods has brought significant contribution over the years, but innovative methods are quickly emerging and allow more in-depth understanding of the mechanisms, aside from a simple quantification. Due to increased sensitivity, time efficiency, pathway specificity and negligible cytotoxicity, these innovative approaches have great potential for both in vitro and in vivo studies. This review aims to shed light on the importance of developing and using novel nanoscale methods as an alternative to the classic apoptosis detection techniques.

Facile Microwave Assisted Synthesis of Silver Nanostars for Ultrasensitive Detection of Biological Analytes by SERS.

We report a very simple, rapid and reproducible method for the fabrication of anisotropic silver nanostars (AgNS) that can be successfully used as highly efficient SERS substrates for different bioanalytes, even in the case of a near-infra-red (NIR) excitation laser. The nanostars have been synthesized using the chemical reduction of Ag+ ions by trisodium citrate. This is the first research reporting the synthesis of AgNS using only trisodium citrate as a reducing and stabilizing agent. The key elements of this original synthesis procedure are rapid hydrothermal synthesis of silver nanostars followed by a cooling down procedure by immersion in a water bath. The synthesis was performed in a sealed bottom flask homogenously heated and brought to a boil in a microwave oven. After 60 s, the colloidal solution was cooled down to room temperature by immersion in a water bath at 35 °C. The as-synthesized AgNS were washed by centrifugation and used for SERS analysis of test molecules (methylene blue) as well as biological analytes: pharmaceutical compounds with various Raman cross sections (doxorubicin, atenolol & metoprolol), cell lysates and amino acids (methionine & cysteine). UV-Vis absorption spectroscopy, (Scanning) Transmission Electron Microscopy ((S)TEM) and Atomic Force Microscopy (AFM) have been employed for investigating nanostars' physical properties.

Hybrid Lipid Nanoformulations for Hepatoma Therapy: Sorafenib Loaded Nanoliposomes-A Preliminary Study.

Sorafenib is a multikinase inhibitor that has received increasing attention due to its high efficacy in hepatocellular carcinoma treatment. However, its poor pharmacokinetic properties (limited water solubility, rapid elimination, and metabolism) still represent major bottlenecks that need to be overcome in order to improve Sorafenib's clinical application. In this paper, we propose a nanotechnology-based hybrid formulation that has the potential to overcome these challenges: sorafenib-loaded nanoliposomes. Sorafenib molecules have been incorporated into the hydrophobic lipidic bilayer during the synthesis process of nanoliposomes using an original procedure developed in our laboratory and, to the best of our knowledge, this is the first paper reporting this type of analysis. The liposomal hybrid formulations have been characterized by transmission electron microscopy (TEM), dynamic light scattering (DLS), and nanoparticle tracking analysis (NTA) that provided useful information concerning their shape, size, zeta-potential, and concentration. The therapeutic efficacy of the nanohybrids has been evaluated on a normal cell line (LX2) and two hepatocarcinoma cell lines, SK-HEP-1 and HepG2, respectively.

Targeting Cell Death Mechanism Specifically in Triple Negative Breast Cancer Cell Lines.

Triple negative breast cancer (TNBC) is currently associated with a lack of treatment options. Arsenic derivatives have shown antitumoral activity both in vitro and in vivo; however, their mode of action is not completely understood. In this work we evaluate the response to arsenate of the double positive MCF-7 breast cancer cell line as well as of two different TNBC cell lines, Hs578T and MDA-MB-231. Multimodal experiments were conducted to this end, using functional assays and microarrays. Arsenate was found to induce cytoskeletal alteration, autophagy and apoptosis in TNBC cells, and moderate effects in MCF-7 cells. Gene expression analysis showed that the TNBC cell lines' response to arsenate was more prominent in the G2M checkpoint, autophagy and apoptosis compared to the Human Mammary Epithelial Cells (HMEC) and MCF-7 cell lines. We confirmed the downregulation of anti-apoptotic genes (MCL1, BCL2, TGFß1 and CCND1) by qRT-PCR, and on the protein level, for TGFß2, by ELISA. Insight into the mode of action of arsenate in TNBC cell lines it is provided, and we concluded that TNBC and non-TNBC cell lines reacted differently to arsenate treatment in this particular experimental setup. We suggest the future research of arsenate as a treatment strategy against TNBC.

A Screening Study for the Development of Simvastatin-Doxorubicin Liposomes, a Co-Formulation with Future Perspectives in Colon Cancer Therapy.

An increasing number of studies published so far have evidenced the benefits of Simvastatin (SIM) and Doxorubicin (DOX) co-treatment in colorectal cancer. In view of this, the current study aimed to investigate the pharmaceutical development of liposomes co-encapsulating SIM and DOX, by implementing the Quality by Design (QbD) concept, as a means to enhance the antiproliferative effect of the co-formulation on C26 murine colon cancer cells co-cultured with macrophages. It is known that the quality profile of liposomes is dependent on the critical quality attributes (CQAs) of liposomes (drug entrapped concentration, encapsulation efficiency, size, zeta potential, and drug release profile), which are, in turn, directly influenced by various formulation factors and processing parameters. By using the design of experiments, it was possible to outline the increased variability of CQAs in relation to formulation factors and identify by means of statistical analysis the material attributes that are critical (phospholipids, DOX and SIM concentration) for the quality of the co-formulation. The in vitro studies performed on a murine colon cancer cell line highlighted the importance of delivering the optimal drug ratio at the target site, since the balance antiproliferative vs. pro-proliferative effects can easily be shifted when the molar ratio between DOX and SIM changes.

Quantifying Cytosolic Cytochrome c Concentration Using Carbon Quantum Dots as a Powerful Method for Apoptosis Detection.

BACKGROUND: Cytochrome c (Cyt c) is a key biomarker for early apoptosis, and many methods were designed to detect its release from mitochondria. For a proper evaluation of these programed cell death mechanisms, fluorescent nanoparticles are excellent candidates due to their valuable optical properties. Among all classes of nanoparticles developed thus far, carbon-based quantum dots bring qualitative and efficient imaging strategies for biomedical applications as a consequence of their biocompatibility and low cytotoxicity. METHODS: In this study, we synthesized carbon quantum dots smaller than 5 nm from sodium citrate and polyethylene imine. These nanoparticles were rigorously characterized, and their quenching capacity in apoptotic events was assessed in A549 cells treated with staurosporine and etoposide. For the evaluation of Cyt c release, a phenomenon directly correlated with apoptotic events, we ran a semiquantitative analysis using confocal laser scanning microscopy. RESULTS: Carbon quantum dots were synthesized and were successfully employed for Cyt c detection by means of fluorescence microscopy. Significant drops in fluorescence intensity were observed in the case of cells treated with apoptosis-inducing therapeutic compounds compared to untreated cells, confirming Cyt c release from mitochondria to cytosol. CONCLUSION: Considering these results, we strongly believe this method can contribute to an indirect in vitro evaluation of apoptosis.

Hsa-miR-125b Therapeutic Role in Colon Cancer Is Dependent on the Mutation Status of the TP53 Gene.

Colon cancer is the third most common cancer type worldwide and is highly dependent on DNA mutations that progressively appear and accumulate in the normal colon epithelium. Mutations in the TP53 gene appear in approximately half of these patients and have significant implications in disease progression and response to therapy. miR-125b-5p is a controversial microRNA with a dual role in cancer that has been reported to target specifically TP53 in colon adenocarcinomas. Our study investigated the differential therapeutic effect of miR-125b-5p replacement in colon cancer based on the TP53 mutation status of colon cancer cell lines. In TP53 mutated models, miR-125b-5p overexpression slows cancer cells' malignant behavior by inhibiting the invasion/migration and colony formation capacity via direct downregulation of mutated TP53. In TP53 wild type cells, the exogenous modulation of miR-125b-5p did not significantly affect the molecular and phenotypic profile. In conclusion, our data show that miR-125b-5p has an anti-cancer effect only in TP53 mutated colon cancer cells, explaining partially the dual behavior of this microRNA in malignant pathologies.

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.

New perspectives in triple-negative breast cancer therapy based on treatments with TGFß1 siRNA and doxorubicin.

Triple-negative breast cancer (TNBC), which accounts for 10-20% of all breast cancers, has the worst prognosis. Although chemotherapy treatment is a standard for TNBC, it lacks a specific target. Therefore, new therapeutic strategies are required to be investigated. In this study, a combined doxorubicin (DOX) and small interfering RNA (siRNA) therapy is proposed as therapeutic strategy for targeting TGFß1 gene. Hs578T cell line is used as in vitro model for TNBC, wherein TGFß1siRNA therapy is employed to enhance therapeutic effects. Cell proliferation rate is measured using an MTT test, and morphological alterations are assed using microscopically approached, while gene expression is determined by qRT-PCR analysis. The combined treatment of TGFß1siRNA and DOX reduced levels of cell proliferation and mitochondrial activity and promoted the alteration of cell morphology (dark-field microscopy). DOX treatment caused downregulation of six genes and upregulation of another six genes. The combined effects of DOX and TGFß1siRNA resulted in upregulation of 13 genes and downregulation of four genes. Silencing of TGFß1 resulted in activation of cell death mechanisms in Hs578T cells, to potentiate the effects of DOX, but not in an additive manner, due to the activation of genes involved in resistance to therapy (ABCB1 and IL-6).

Hyperthermia, Cytotoxicity, and Cellular Uptake Properties of Manganese and Zinc Ferrite Magnetic Nanoparticles Synthesized by a Polyol-Mediated Process.

Manganese and zinc ferrite magnetic nanoparticles (MNPs) were successfully synthesizedusing the polyol method in ethylene glycol and were found to have high saturation magnetizationvalues (90-95 emu/g at 4 K) when formed by ~30-nm crystallites assembled in an ~80-nm multicorestructure. Hyperthermia data revealed a sigmoidal dependence of the specific absorption rate (SAR)on the alternating magnetic field (AMF) amplitude, with remarkable saturation SAR values in waterof ~1200 W/gFe+Mn and ~800 W/gFe+Zn for the Mn and Zn ferrites, respectively. The immobilizationof the MNPs in a solid matrix reduced the maximum SAR values by ~300 W/gFe+Mn, Zn for bothferrites. The alignment of the MNPs in a uniform static magnetic field, before their immobilizationin a solid matrix, significantly increased their heating performance. Toxicity assays performed infour cell lines revealed a lower toxicity for the Mn ferrites, while in the case of the Zn ferrites, only~50% of cells were viable upon their incubation for 24 h with 0.2 mg/mL of MNPs. Cellular uptakeexperiments revealed that both MNPs entered the cells in a time-dependent manner, as they werefound initially in endosomes and later in the cytosol. All of the studied cell lines were more sensitiveto the ZnFe2O4 MNPs.

Hydrogels Based Drug Delivery Synthesis, Characterization and Administration.

Hydrogels represent 3D polymeric networks specially designed for various medical applications. Due to their porous structure, they are able to swollen and to entrap large amounts of therapeutic agents and other molecules. In addition, their biocompatibility and biodegradability properties, together with a controlled release profile, make hydrogels a potential drug delivery system. In vivo studies have demonstrated their effectiveness as curing platforms for various diseases and affections. In addition, the results of the clinical trials are very encouraging and promising for the use of hydrogels as future target therapy strategies.

The Effects of Low-Dose Irradiation on Human Saliva: A Surface-Enhanced Raman Spectroscopy Study.

Biological effects of low-dose ionizing radiation (IR) have been unclear until now. Saliva, because of the ease of collection, could be valuable in studying low-dose IR effects by means of surface-enhanced Raman spectroscopy (SERS). The objective of our study was to compare the salivary SER spectra recorded before and after low-dose IR exposure in the case of pediatric patients (PP). Unstimulated saliva was collected from ten PP before and after irradiation with a cone beam computed tomography (CBCT) machine used for diagnostic purposes. The SERS measurements have been recorded on dried saliva samples using a solid nanosilver plasmonic substrate synthesized using an original method developed in our laboratory. The experimental results showed that salivary SER spectra are dominated by three vibrational bands (441,735 and 2107 cm-1) that can be assigned to bending and stretching vibrations of salivary thiocyanate (SCN-). After exposure, an immediate increase of vibrational bands assigned to SCN- has been recorded in the case of all samples, probably as a result of IR interaction with oral cavity. This finding suggests that SCN- could be used as a valuable biomarker for the detection and identification of low-dose radiation effects.

Gold nanorods: from anisotropy to opportunity. An evolution update.

Gold nanoparticles have drawn attention to nanomedicine for many years due to their physicochemical properties, which include: good stability; biocompatibility; easy surface chemistry and superior magnetic; and last, electronic properties. All of these properties distinguish gold nanoparticles as advantageous carriers to be exploited. The challenge to develop new gold nanostructures has led to anisotropy, a new property to exploit for various medical applications: diagnostic and imaging strategies as well as therapeutic options. Gold nanorods are the most studied anisotropic gold nanoparticles because of the presence of two absorption peaks according to their longitudinal and transversal plasmon resonances. The longitudinal surface plasmonic resonance can provide the absorption in the near-infrared region and this is an important aspect of using gold nanorods for medical purposes.

Inhibitory Effect of CAPE and Kaempferol in Colon Cancer Cell Lines-Possible Implications in New Therapeutic Strategies.

BACKGROUND: Phytochemicals are natural compounds synthesized as secondary metabolites in plants and represent an important source of molecules with therapeutic applications. Attention is accorded to their potential in anti-cancer therapies as single agents or adjuvant treatment. Herby, we evaluated the in vitro effects of a panel of natural compounds with focus on caffeic acid phenethyl ester (CAPE) and Kaempferol for the treatment of human colon cancer. METHODS: We exposed two human colon cancer cell lines, RKO and HCT-116, followed by functional examination of cell viability, cell proliferation and invasion, cell cycle, apoptosis, and autophagy. Modifications in gene expression were investigated through microarray and detection of existing mutations and finding of new ones was done with the help of Next Generation Sequencing (NGS). RESULTS: Both CAPE and Kaempferol inhibit cell proliferation, motility and invasion, and stimulate apoptosis and autophagy, concomitant with modifications in coding and noncoding genes' expression. Moreover, there are pathogenic mutations that are no longer found upon treatment with CAPE and Kaempferol. CONCLUSIONS: Our findings indicate that CAPE and Kaempferol have the ability to negatively influence the development and advancement of colon cancer in vitro by specifically altering the cells at the molecular level; this activity can be exploited in possible adjuvant therapies once the optimal dose concentration with minimal side effects but with cancer inhibitory activity is set in vivo.

Aberrant miRNAs expressed in HER-2 negative breast cancers patient.

BACKGROUND: Breast cancer is a highly heterogeneous pathology, exhibiting a number of subtypes commonly associated with a poor outcome. Due to their high stability, microRNAs are often regarded as non-invasive cancer biomarkers, having an expression pattern specific for their 'cell of origin'. METHOD: Triple negative breast cancer (TNBC: ER-, PR-, Her-2-) and double positive breast cancer (DPBC: ER+, PR+, Her-2) miRNA expression patterns were obtained by analysis of the TCGA (The Cancer Genome Atlas) data, followed by PCR-array analysis on plasma samples from 20 TNBC patients, 14 DPBC patients and 11 controls. RESULTS: Three downregulated and nine upregulated miRNAs were obtained from the TNBC analysis. Five overexpressed miRNAs were identified in the DPBC group. Four of the dysregulated miRNAs (miR-10a, miR-125b, miR-210 and miR-489) were common for both groups. The cluster miR-17-92 (miR-17, miR-20a, miR-20b, and miR-93), along with miR-130, miR-22 and miR-29a/c, were found to differentiate between TNBC and DPBC. A panel of five transcripts (miR-10a, miR-125, miR-193b, miR-200b and miR-489) was validated in a new set of plasma samples. The overlapping of TCGA and plasma profiling data revealed miR-200b, miR-200c, miR-210 and miR-29c as common signature. MiR-200b was validated on additional normal and tumor tissue samples. The expression level of this transcript from the TCGA data was correlated with lung and bone metastatic genes. CONCLUSION: The miR-200b presents a great potential for the future advancements in the diagnostic/prognostic and therapeutic approach of TNBC, along with other coding or non-coding transcripts. However, this needs to be further integrated in a regulatory network that acts in conjunction with other markers that affect the patients' prognosis or response to therapy.

Exosome-carried microRNA-based signature as a cellular trigger for the evolution of chronic lymphocytic leukemia into Richter syndrome.

Even if considered a cumulative and not a proliferative CD5+ B-cell neoplasm, chronic lymphocytic leukemia (CLL) has a proliferation rate higher than that recognized earlier, especially in the lymphoid tissues. Some patients with CLL develop a clinical syndrome entitled Richter syndrome (RS). Understanding CLL genetics and epigenetics may help to elucidate the molecular basics of the clinical heterogeneity of this type of malignancy. In the present project we aimed to identify a microRNA species that can predict the evolution of therapy-resistant CLL towards RS. In the first phase of our study, microRNA-19b was identified as a possible target, and in the second phase, we transfected three different CLL cell lines with microRNA-19b mimic and inhibitor and assessed the potential role on leukemia cells in vitro. The mechanism by which miR-19b acts were identified as the upregulation of Ki67 and downregulation of p53. This was further supported through RT-PCR and western blotting on CLL cell lines, as well as by next generation sequencing on two patients diagnosed with CLL that evolved into RS.

The silent healer: miR-205-5p up-regulation inhibits epithelial to mesenchymal transition in colon cancer cells by indirectly up-regulating E-cadherin expression.

EMT represents the dominant program within advanced stages of colon cancer, where cells acquire migratory characteristics in order to invade secondary tissues and form metastasis. Where the majority of the therapeutic strategies are concentrated on the reduction of the tumor mass through different apoptotic mechanisms, the present study advocates an important role for miR-205-5p in impairment of colon cancer cells migration and restoration of the epithelial phenotype. Upon identification of a homogenous downregulated profile for miR-205-5p in colon adenocarcinoma patients, functional studies demonstrated that experimental upregulation of this sequence is able to significantly raise the levels of E-cadherin through direct inhibition of ZEB1. Moreover, the elevation in CDH1 expression was translated into functional parameters where cells lost their invasion and migratory characteristics and formed homogenous clusters through adhesion interactions. Survival analysis of colon adenocarcinoma patients revealed that low levels of miR-205-5p are associated with an unfavorable prognostic compared to those with increased expression, demonstrating the possible clinical utility of miR-205-5p replacement. Exogenous administration of miRNA mimics was not associated with significant changes in cell viability or inflammatory pathways. Therefore, the proposed strategy is aiming towards inhibition of metastasis and limitation of the tumor borders in advanced stages patients in order to prolong the survival time and to increase the efficiency of the current therapeutic strategies.

Small versus Large Iron Oxide Magnetic Nanoparticles: Hyperthermia and Cell Uptake Properties.

Efficient use of magnetic hyperthermia in clinical cancer treatment requires biocompatible magnetic nanoparticles (MNPs), with improved heating capabilities. Small (~34 nm) and large (~270 nm) Fe3O4-MNPs were synthesized by means of a polyol method in polyethylene-glycol (PEG) and ethylene-glycol (EG), respectively. They were systematically investigated by means of X-ray diffraction, transmission electron microscopy and vibration sample magnetometry. Hyperthermia measurements showed that Specific Absorption Rate (SAR) dependence on the external alternating magnetic field amplitude (up to 65 kA/m, 355 kHz) presented a sigmoidal shape, with remarkable SAR saturation values of ~1400 W/gMNP for the small monocrystalline MNPs and only 400 W/gMNP for the large polycrystalline MNPs, in water. SAR values were slightly reduced in cell culture media, but decreased one order of magnitude in highly viscous PEG1000. Toxicity assays performed on four cell lines revealed almost no toxicity for the small MNPs and a very small level of toxicity for the large MNPs, up to a concentration of 0.2 mg/mL. Cellular uptake experiments revealed that both MNPs penetrated the cells through endocytosis, in a time dependent manner and escaped the endosomes with a faster kinetics for large MNPs. Biodegradation of large MNPs inside cells involved an all-or-nothing mechanism.