Exosomes secreted from human colon cancer cells influence the adhesion of neighboring metastatic cells: Role of microRNA-210

Cancer-secreted exosomes influence tumor microenvironment and support cancer growth and metastasis. MiR-210 is frequently up-regulated in colorectal cancer tissues and correlates with metastatic disease. We investigated whether exosomes are actively released by HCT-8 colon cancer cells, the role of exosomal miR-210 in the cross-talk between primary cancer cells and neighboring metastatic cells and its contribution in regulating epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET). After 7 d of culture, a subpopulation of viable HCT-8 cells detached the monolayer and started to grow in suspension, suggesting anoikis resistance and a metastatic potential. The expression of key proteins of EMT revealed that these cells were E-cadherin negative and vimentin positive further confirming their metastatic phenotype and the acquisition of anoikis resistance. Metastatic cells, in the presence of adherently growing HCT-8, continued to grow in suspension whereas only if seeded in cell-free wells, were able to adhere again and to form E-cadherin positive and vimentin negative new colonies, suggesting the occurrence of MET. The chemosensitivity to 5 fluorouracil and to FOLFOX-like treatment of metastatic cells was significantly diminished compared to adherent HCT-8 cells. Of note, adherent new colonies undergoing MET, were insensitive to both chemotherapeutic strategies. Electron microscopy analysis demonstrated that adherently growing HCT-8, actually secreted exosomes and that exosomes in turn were taken up by metastatic cells. When exosomes secreted by adherently growing HCT-8 were administered to metastatic cells, MET was significantly inhibited. miR-210 was significantly upregulated in exosomes compared to its intracellular levels in adherently growing HCT-8 cells and correlated to anoikis resistance and EMT markers. Exosomes containing miR-210 might be considered as EMT promoting signals that preserve the local cancer-growth permissive milieu and also guide metastatic cells to free, new sites of dissemination

Oxaliplatin Neurotoxicity Involves Peroxisome Alterations. PPARc Agonism as Preventive Pharmacological Approach

The development of neuropathic syndromes is an important, dose limiting side effect of anticancer agents like platinum derivates, taxanes and vinca alkaloids. The causes of neurotoxicity are still unclear but the impairment of the oxidative equilibrium is strictly related to pain. Two intracellular organelles, mitochondria and peroxisomes cooperate to the maintaining of the redox cellular state. Whereas a relationship between chemotherapy-dependent mitochondrial alteration and neuropathy has been established, the role of peroxisome is poor explored. In order to study the mechanisms of oxaliplatin-induced neurotoxicity, peroxisomal involvement was evaluated in vitro and in vivo. In primary rat astrocyte cell culture, oxaliplatin (10 µM for 48 h or 1 µM for 5 days) increased the number of peroxisomes, nevertheless expression and functionality of catalase, the most important antioxidant defense enzyme in mammalian peroxisomes, were significantly reduced. Five day incubation with the selective Peroxisome Proliferator Activated Receptor-γ (PPAR-γ) antagonist G3335 (30 µM) induced a similar peroxisomal impairment suggesting a relationship between PPARγ signaling and oxaliplatin neurotoxicity. The PPARγ agonist rosiglitazone (10 µM) reduced the harmful effects induced both by G3335 and oxaliplatin. In vivo, in a rat model of oxaliplatin induced neuropathy, a repeated treatment with rosiglitazone (3 and 10 mg kg(-1) per os) significantly reduced neuropathic pain evoked by noxious (Paw pressure test) and non-noxious (Cold plate test) stimuli. The behavioral effect paralleled with the prevention of catalase impairment induced by oxaliplatin in dorsal root ganglia. In the spinal cord, catalase protection was showed by the lower rosiglitazone dosage without effect on the astrocyte density increase induced by oxaliplatin. Rosiglitazone did not alter the oxaliplatin-induced mortality of the human colon cancer cell line HT-29. These results highlight the role of peroxisomes in oxaliplatin-dependent nervous damage and suggest PPARγ stimulation as a candidate to counteract oxaliplatin neurotoxicity

Ethyl acetate extract from Cistus x incanus L. leaves enriched in myricetin and quercetin derivatives, inhibits inflammatory mediators and activates Nrf2/HO-1 pathway in LPS-stimulated RAW 264.7 macrophages.

Abstract Cistus x incanus L. is a Mediterranean evergreen shrub used in folk medicine for the treatment of inflammatory disorders but the underlying mechanisms are not fully understood. We therefore investigated the anti-inflammatory effects of an ethyl acetate fraction (EAF) from C. x incanus L. leaves on lipopolysaccharide (LPS) activated RAW 264.7 macrophages. HPLC analysis revealed myricetin and quercetin derivatives to be the major compounds in EAF; EAF up to 1 µM of total phenolic content, was not cytotoxic and inhibited the mRNA expression of interleukin-6 (IL-6) and cyclooxygenase-2 (COX-2) (p < 0.05) and the production of prostaglandins E2 (PGE2) (p < 0.05). Meanwhile, EAF triggered the mRNA expression of interleukin-10 (IL-10) and elicited the nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2), as well as the expression of its main target gene, heme oxygenase-1 (HO-1) (p < 0.05). These data indicate that EAF attenuates experimental inflammation via the inhibition of proinflammatory mediators and at least in part, by the activation of Nrf2/HO-1 pathway. These effects are likely due to myricetin and quercetin derivatives but the role of other, less abundant components cannot be excluded. Further studies to confirm the relevance of our findings in animal models and to highlight the relative contribution of each component to the anti-inflammatory activity of EAF should be conducted

The Anti-Arthritic Efficacy of Khellin Loaded in Ascorbyl Decanoate Nanovesicles after an Intra-Articular Administration

Osteoarthritis is the most widespread joint-affecting disease. The management of persistent pain remains inadequate and demands new therapeutic strategies. In this study, we explored the pain relieving and protective properties of a single intra-articular (i.a.) injection of khellin loaded in nanovesicles (K-Ves) based on ascorbyl decanoate plus phosphatidylcholine in a rat model of osteoarthritis (OA) induced by monosodium iodoacetate (MIA) treatment. The developed nanovesicles (approximately 136 nm) had a narrow size distribution (PdI 0.26), a good recovery (about 80%) and a worthy encapsulation efficiency (about 70%) with a ζ-potential of about −40 mV. The stability of K-Ves was assessed in simulated synovial fluid. Seven days after the articular damage with MIA, both K-Ves and a suspension of khellin (K, 50 μL) were i.a. injected. K-Ves significantly counteracted MIA-induced hypersensitivity to mechanical noxious (paw pressure test) and non-noxious stimuli (von Frey test) and significantly reduced the postural unbalance related to spontaneous pain (incapacitance test) and the motor alterations (beam balance test) 7 and 14 days after the i.a. injection. K was partially active only on day 7 after the treatment. The histology emphasized the improvement of several morphological factors in MIA plus K-Ves-treated animals. In conclusion, K-Ves could be successfully used for the local treatment of osteoarthritis

Formulation of Nanomicelles to Improve the Solubility and the Oral Absorption of Silymarin

Two novel nanomicellar formulations were developed to improve the poor aqueous solubility and the oral absorption of silymarin. Polymeric nanomicelles made of Soluplus and mixed nanomicelles combining Soluplus with d-&#945;-tocopherol polyethylene glycol 1000 succinate (vitamin E TPGS) were prepared using the thin film method. Physicochemical parameters were investigated, in particular the average diameter, the homogeneity (expressed as polydispersity index), the zeta potential, the morphology, the encapsulation efficiency, the drug loading, the critical micellar concentration and the cloud point. The sizes of ~60 nm, the narrow size distribution (polydispersity index &#8804;0.1) and the encapsulation efficiency &gt;92% indicated the high affinity between silymarin and the core of the nanomicelles. Solubility studies demonstrated that the solubility of silymarin increased by ~6-fold when loaded into nanomicelles. Furthermore, the physical and chemical parameters of SLM-loaded formulations stored at room temperature and in refrigerated conditions (4 &#176;C) were monitored over three months. In vitro stability and release studies in media miming the physiological conditions were also performed. In addition, both formulations did not alter the antioxidant properties of silymarin as evidenced by the 1,1-Diphenyl-2-picrylhydrazyl radical (DPPH) assay. The potential of the nanomicelles to increase the intestinal absorption of silymarin was firstly investigated by the parallel artificial membrane permeability assay. Subsequently, transport studies employing Caco-2 cell line demonstrated that mixed nanomicelles statistically enhanced the permeability of silymarin compared to polymeric nanomicelles and unformulated extract. Finally, the uptake studies indicated that both nanomicellar formulations entered into Caco-2 cells via energy-dependent mechanisms

Thyroid Hormone, Thyroid Hormone Metabolites and Mast Cells: A Less Explored Issue

Mast cells are primary players in immune and inflammatory diseases. In the brain, mast cells are located at the brain side of the blood brain barrier (BBB) exerting a crucial role in protecting the brain from xenobiotic invasion. Furthermore, recent advances in neuroscience indicate mast cells may play an important role in glial cell-neuron communication through the release of mediators, including histamine. Interestingly, brain mast cells contain not only 50% of the brain histamine but also hormones, proteases and lipids or amine mediators; and cell degranulation may be triggered by different stimuli activating membrane bound receptors including the four types of histaminergic receptors. Among hormones, mast cells can store thyroid hormone (T3) and express membrane-bound thyroid stimulating hormone receptors (TSHRs), thus suggesting from one side that thyroid function may affect mast cells function, from the other that mast cell degranulation may impact on thyroid function. In this respect, the research on hormones in mast cells is scarce. Recent pharmacological evidence indicates the existence of a non-genomic portion of the thyroid secretion including thyroid hormone metabolites. Among which the 3,5 diiodothyronine (3,5-T2), 3-iodothyroanamine (T1AM) and 3-iodothyroacetic acid (TA1) are the most studied. All these compounds are endogenously occurring and found to be increased in inflammatory-based diseases involving mast cells. T1AM and TA1 induce, as T3, neuroprotective effects and itch but also hyperalgesia in rodents with a mechanism largely unknown but mediated by the release of histamine. Due to the rapid onset of their effectiveness they may trigger histamine release from a cell where it is “ready-to-be released,” i.e., mast cells. Following a very thin path which passes through old experimental and clinical evidence, at the light of novel acquisitions on endogenous T3 metabolites, we aim to stimulate the attention on the possibility that mast cell histamine may be the connector of a novel (neuro) endocrine pathway linking the thyroid with mast cells

Development and characterization of an in vitro model of colorectal adenocarcinoma with MDR phenotype

The major cause of failure in cancer chemotherapy is the development of multidrug resistance (MDR), and the characterization of biological factors involved in this response to therapy is particularly needed. A doxorubicin‐resistant HCT‐8/R clone was selected from sensitive parental cells and characterized analyzing several parameters (cell cycle phase distribution, apoptotic activity, expression, distribution and functionality of the P‐gp efflux pump, the response to other chemotherapy agents, its ultrastructural features, invasiveness, and transcriptomic profile). HCT‐8/R cells showed a peculiar S phase distribution, characterized by a single pulse of proliferation, resistance to drug‐mediated apoptosis, increased expression and functionality of P‐gp and overexpression of stem cell markers (CD44 and aldehyde dehydrogenase 1A2). At the ultrastructural level, HCT‐8/R presented a greater cell volume and several intracytoplasmic vesicles respect to HCT‐8. Moreover, the resistant clone was characterized by cross resistance to other cytotoxic drugs and a greater capacity for migration and invasion, compared to parental cells. Our data reinforce the concept that the MDR phenotype in HCT‐8/R cells is multifactorial and involves multiple mechanisms, representing an interesting tool to understand the biological basis of MDR and to test strategies that overcome resistance to chemotherapy