Colorectal Cancer Cell Invasion and Functional Properties Depend on Peri-Tumoral Extracellular Matrix.

We investigated how the extracellular matrix (ECM) affects LoVo colorectal cancer cells behavior during a spatiotemporal invasion. Epithelial-to-mesenchymal transition (EMT) markers, matrix-degrading enzymes, and morphological phenotypes expressed by LoVo-S (doxorubicin-sensitive) and higher aggressive LoVo-R (doxorubicin-resistant) were evaluated in cells cultured for 3 and 24 h on Millipore filters covered by Matrigel, mimicking the basement membrane, or type I Collagen reproducing a desmoplastic lamina propria. EMT and invasiveness were investigated with RT-qPCR, Western blot, and scanning electron microscopy. As time went by, most gene expressions decreased, but in type I Collagen samples, a strong reduction and high increase in MMP-2 expression in LoVo-S and -R cells occurred, respectively. These data were confirmed by the development of an epithelial morphological phenotype in LoVo-S and invading phenotypes with invadopodia in LoVo-R cells as well as by protein-level analysis. We suggest that the duration of culturing and type of substrate influence the morphological phenotype and aggressiveness of both these cell types differently. In particular, the type I collagen meshwork, consisting of large fibrils confining inter fibrillar micropores, affects the two cell types differently. It attenuates drug-sensitive LoVo-S cell aggressiveness but improves a proteolytic invasion in drug-resistant LoVo-R cells as time goes by. Experimental studies on CRC cells should examine the peri-tumoral ECM components, as well as the dynamic physical conditions of TME, which affect the behavior and aggressiveness of both drug-sensitive and drug-resistant LoVo cells differently.

Shedding Light on Osteosarcoma Cell Differentiation: Impact on Biomineralization and Mitochondria Morphology.

Osteosarcoma (OS) is the most common primary malignant bone tumor and its etiology has recently been associated with osteogenic differentiation dysfunctions. OS cells keep a capacity for uncontrolled proliferation showing a phenotype similar to undifferentiated osteoprogenitors with abnormal biomineralization. Within this context, both conventional and X-ray synchrotron-based techniques have been exploited to deeply characterize the genesis and evolution of mineral depositions in a human OS cell line (SaOS-2) exposed to an osteogenic cocktail for 4 and 10 days. A partial restoration of the physiological biomineralization, culminating with the formation of hydroxyapatite, was observed at 10 days after treatment together with a mitochondria-driven mechanism for calcium transportation within the cell. Interestingly, during differentiation, mitochondria showed a change in morphology from elongated to rounded, indicating a metabolic reprogramming of OS cells possibly linked to an increase in glycolysis contribution to energy metabolism. These findings add a dowel to the genesis of OS giving new insights on the development of therapeutic strategies able to restore the physiological mineralization in OS cells.

Substrate Type and Concentration Differently Affect Colon Cancer Cells Ultrastructural Morphology, EMT Markers, and Matrix Degrading Enzymes.

Aim of the study was to understand the behavior of colon cancer LoVo-R cells (doxorubicin-resistant) vs. LoVo-S (doxorubicin sensitive) in the initial steps of extracellular matrix (ECM) invasion. We investigated how the matrix substrates Matrigel and type I collagen-mimicking the basement membrane (BM) and the normal or desmoplastic lamina propria, respectively-could affect the expression of epithelial-to-mesenchymal transition (EMT) markers, matrix-degrading enzymes, and phenotypes. Gene expression with RT-qPCR, E-cadherin protein expression using Western blot, and phenotypes using scanning electron microscopy (SEM) were analyzed. The type and different concentrations of matrix substrates differently affected colon cancer cells. In LoVo-S cells, the higher concentrated collagen, mimicking the desmoplastic lamina propria, strongly induced EMT, as also confirmed by the expression of Snail, metalloproteases (MMPs)-2, -9, -14 and heparanase (HPSE), as well as mesenchymal phenotypes. Stimulation in E-cadherin expression in LoVo-S groups suggests that these cells develop a hybrid EMT phenotype. Differently, LoVo-R cells did not increase their aggressiveness: no changes in EMT markers, matrix effectors, and phenotypes were evident. The low influence of ECM components in LoVo-R cells might be related to their intrinsic aggressiveness related to chemoresistance. These results improve understanding of the critical role of tumor microenvironment in colon cancer cell invasion, driving the development of new therapeutic approaches.

Development and Characterization of Azithromycin-Loaded Microemulsions: A Promising Tool for the Treatment of Bacterial Skin Infections.

In recent years, the treatment of bacterial skin infections has been considered a major healthcare issue due to the growing emergence of antibiotic-resistant strains of Staphylococcus aureus. The incorporation of antibiotics in appropriate nanosystems could represent a promising strategy, able to overcome several drawbacks of the topical treatment of infections, including poor drug retention within the skin. The present work aims to develop microemulsions containing azithromycin (AZT), a broad-spectrum macrolide antibiotic. Firstly, AZT solubility in various oils, surfactants and co-surfactants was assessed to select the main components. Subsequently, microemulsions composed of vitamin E acetate, Labrasol® and Transcutol® P were prepared and characterized for their pH, viscosity, droplet size, zeta potential and ability to release the drug and to promote its retention inside porcine skin. Antimicrobial activity against S. aureus methicillin-resistant strains (MRSA) and the biocompatibility of microemulsions were evaluated. Microemulsions showed an acceptable pH and were characterized by different droplet sizes and viscosities depending on their composition. Interestingly, they provided a prolonged release of AZT and promoted its accumulation inside the skin. Finally, microemulsions retained AZT efficacy on MRSA and were not cytotoxic. Hence, the developed AZT-loaded microemulsions could be considered as useful nanocarriers for the treatment of antibiotic-resistant infections of the skin.

Natural-like Chalcones with Antitumor Activity on Human MG63 Osteosarcoma Cells.

Osteosarcoma (OS) is a malignant disease characterized by poor prognosis due to a high incidence of metastasis and chemoresistance. Recently, Licochalcone A (Lic-A) has been reported as a promising agent against OS. Starting from chalcones selected from a wide in-house library, a new series was designed and synthetized. The antitumor activity of the compounds was tested on the MG63 OS cell line through the innovative Quantitative Phase Imaging technique and MTT assay. To further investigate the biological profile of active derivatives, cell cycle progression and apoptosis induction were evaluated. An earlier and more consistent arrest in the G2-M phase with respect to Lic-A was observed. Moreover, apoptosis was assessed by Annexin V staining as well as by the detection of typical morphological features of apoptotic cells. Among the selected compounds, 1e, 1q, and 1r proved to be the most promising antitumor molecules. This study pointed out that an integrated methodological approach may constitute a valuable platform for the rapid screening of large series of compounds.

Development of Spanish Broom and Flax Dressings with Glycyrrhetinic Acid-Loaded Films for Wound Healing: Characterization and Evaluation of Biological Properties.

The selection of an appropriate dressing for each type of wound is a very important procedure for a faster and more accurate healing process. So, the aim of this study was to develop innovative Spanish Broom and flax wound dressings, as alternatives to cotton used as control, with polymeric films containing glycyrrhetinic acid (GA) to promote wound-exudate absorption and the healing process. The different wound dressings were prepared by a solvent casting method, and characterized in terms of drug loading, water uptake, and in vitro release. Moreover, biological studies were performed to evaluate their biocompatibility and wound-healing efficacy. Comparing the developed wound dressings, Spanish Broom dressings with GA-loaded sodium hyaluronate film had the best functional properties, in terms of hydration ability and GA release. Moreover, they showed a good biocompatibility, determining a moderate induction of cell proliferation and no cytotoxicity. In addition, the wound-healing test revealed that the Spanish Broom dressings promoted cell migration, further facilitating the closure of the wound.

Calcite as a Precursor of Hydroxyapatite in the Early Biomineralization of Differentiating Human Bone-Marrow Mesenchymal Stem Cells.

Biomineralization is the process by which living organisms generate organized mineral crystals. In human cells, this phenomenon culminates with the formation of hydroxyapatite, which is a naturally occurring mineral form of calcium apatite. The mechanism that explains the genesis within the cell and the propagation of the mineral in the extracellular matrix still remains largely unexplained, and its characterization is highly controversial, especially in humans. In fact, up to now, biomineralization core knowledge has been provided by investigations on the advanced phases of this process. In this study, we characterize the contents of calcium depositions in human bone mesenchymal stem cells exposed to an osteogenic cocktail for 4 and 10 days using synchrotron-based cryo-soft-X-ray tomography and cryo-XANES microscopy. The reported results suggest crystalline calcite as a precursor of hydroxyapatite depositions within the cells in the biomineralization process. In particular, both calcite and hydroxyapatite were detected within the cell during the early phase of osteogenic differentiation. This striking finding may redefine most of the biomineralization models published so far, taking into account that they have been formulated using murine samples while studies in human cell lines are still scarce.

Magnesium: Biochemistry, Nutrition, Detection, and Social Impact of Diseases Linked to Its Deficiency.

Magnesium plays an important role in many physiological functions. Habitually low intakes of magnesium and in general the deficiency of this micronutrient induce changes in biochemical pathways that can increase the risk of illness and, in particular, chronic degenerative diseases. The assessment of magnesium status is consequently of great importance, however, its evaluation is difficult. The measurement of serum magnesium concentration is the most commonly used and readily available method for assessing magnesium status, even if serum levels have no reliable correlation with total body magnesium levels or concentrations in specific tissues. Therefore, this review offers an overview of recent insights into magnesium from multiple perspectives. Starting from a biochemical point of view, it aims at highlighting the risk due to insufficient uptake (frequently due to the low content of magnesium in the modern western diet), at suggesting strategies to reach the recommended dietary reference values, and at focusing on the importance of detecting physiological or pathological levels of magnesium in various body districts, in order to counteract the social impact of diseases linked to magnesium deficiency.

Assessment and Imaging of Intracellular Magnesium in SaOS-2 Osteosarcoma Cells and Its Role in Proliferation.

Magnesium is an essential nutrient involved in many important processes in living organisms, including protein synthesis, cellular energy production and storage, cell growth and nucleic acid synthesis. In this study, we analysed the effect of magnesium deficiency on the proliferation of SaOS-2 osteosarcoma cells. When quiescent magnesium-starved cells were induced to proliferate by serum addition, the magnesium content was 2-3 times lower in cells maintained in a medium without magnesium compared with cells growing in the presence of the ion. Magnesium depletion inhibited cell cycle progression and caused the inhibition of cell proliferation, which was associated with mTOR hypophosphorylation at Serine 2448. In order to map the intracellular magnesium distribution, an analytical approach using synchrotron-based X-ray techniques was applied. When cell growth was stimulated, magnesium was mainly localized near the plasma membrane in cells maintained in a medium without magnesium. In non-proliferating cells growing in the presence of the ion, high concentration areas inside the cell were observed. These results support the role of magnesium in the control of cell proliferation, suggesting that mTOR may represent an important target for the antiproliferative effect of magnesium. Selective control of magnesium availability could be a useful strategy for inhibiting osteosarcoma cell growth.

Multifaceted activity of polyciclic MDR revertant agents in drug-resistant leukemic cells: Role of the spacer.

A small library of derivatives carrying a polycyclic scaffold recently identified by us as a new privileged structure in medicinal chemistry was designed and synthesized, aiming at obtaining potent MDR reverting agents also endowed with antitumor properties. In particular, as a follow-up of our previous studies, attention was focused on the role of the spacer connecting the polycyclic core with a properly selected nitrogen-containing group. A relevant increase in reverting potency was observed, going from the previously employed but-2-ynyl- to a pent-3-ynylamino moiety, as in compounds 3d and 3e, while the introduction of a triazole ring proved to differently impact on the activity of the compounds. The docking results supported the data obtained by biological tests, showing, for the most active compounds, the ability to establish specific bonds with P-glycoprotein. Moreover, a multifaceted anticancer profile and dual in vitro activity was observed for all compounds, showing both revertant and antitumor effects on leukemic cells. In this respect, 3c emerged as a "triple-target" agent, endowed with a relevant reverting potency, a considerable antiproliferative activity and a collateral sensitivity profile.

The assessment of intracellular magnesium: different strategies to answer different questions.

The role of magnesium in cell metabolism is complex and still not completely clarified. Although magnesium has been shown to modulate many phenomena in cells, its intracellular distribution and subcellular compartmentalization have not yet elucidated in detail, mainly as a consequence of the inadequacy of analytical techniques. The method usually employed to quantify total magnesium in cells or tissue are F-AAS or more sensitive techniques as graphite furnace AAS and inductively coupled plasma mass spectroscopy (MS). Thanks to the development of new specific fluorescent dyes, several progresses have been made in the comprehension of the fundamental biological process at the cellular and sub-cellular level. Moreover, the biological function of a chemical element in cells does not only require the determination of its intracellular quantity but also the spatial distribution of its concentration. Most of Mg2+-sensitive fluorescent dyes detect only the free metal ions, precluding the possibility of identifying the total pool of Mg. This review aims at giving an overview on different techniques focusing on two approaches to quantify total Mg in a small cell population or in single cells: i) Indirect Mg detection, label-based methods that represent the best choice to quantify the elemental concentration on a large cell population; ii) direct Mg detection (label-free), Synchrotron-based x-ray microscopy techniques that offer the possibility of achieving a detailed map of the intracellular concentration of a specific chemical element on single cell.

Glycyrrhetinic Acid Liposomes and Hyalurosomes on Spanish Broom, Flax, and Hemp Dressings to Heal Skin Wounds.

The focus of this work was to prepare Spanish Broom, flax, and hemp dressings impregnated with glycyrrhetinic acid (GA) liposomes or hyalurosomes to promote the healing process and protect the skin wounds. Vesicles were prepared by the film hydration method and characterized in terms of size, particle size distribution, ζ potential, encapsulation efficiency, in vitro release, and biocompatibility on 3T3 fibroblasts. Loaded liposomes and hyalurosomes showed nanometric size (355 ± 19 nm and 424 ± 32 nm, respectively), good size distribution (lower than 0.3), and appropriate encapsulation efficiency (58.62 ± 3.25% and 59.22 ± 8.18%, respectively). Hyalurosomes showed good stability during the storage period, which can be correlated to the negative ζ potential, and allowed a fast and complete release of GA. Preliminary biological studies revealed that both kinds of loaded vesicles were not cytotoxic and that hyalurosomes could exert a slight stimulating effect on fibroblast proliferation. Finally, in vitro release studies from the different dressings impregnated with the loaded vesicles demonstrated that a high amount of GA could be reached at the wound site after 60 min from application. In conclusion, the results suggested that the developed dressings, especially those impregnated with hyalurosomes, can be efficiently used to promote the healing process.

Analysis of Intracellular Magnesium and Mineral Depositions during Osteogenic Commitment of 3D Cultured Saos2 Cells.

In this study, we explore the behaviour of intracellular magnesium during bone phenotype modulation in a 3D cell model built to mimic osteogenesis. In addition, we measured the amount of magnesium in the mineral depositions generated during osteogenic induction. A two-fold increase of intracellular magnesium content was found, both at three and seven days from the induction of differentiation. By X-ray microscopy, we characterized the morphology and chemical composition of the mineral depositions secreted by 3D cultured differentiated cells finding a marked co-localization of Mg with P at seven days of differentiation. This is the first experimental evidence on the presence of Mg in the mineral depositions generated during biomineralization, suggesting that Mg incorporation occurs during the bone forming process. In conclusion, this study on the one hand attests to an evident involvement of Mg in the process of cell differentiation, and, on the other hand, indicates that its multifaceted role needs further investigation.

Antitumor Potential and Phytochemical Profile of Plants from Sardinia (Italy), a Hotspot for Biodiversity in the Mediterranean Basin.

Sardinia (Italy), with its wide range of habitats and high degree of endemism, is an important area for plant-based drug discovery studies. In this work, the antitumor activity of 35 samples from Sardinian plants was evaluated on human osteosarcoma cells U2OS. The results showed that five plants were strongly antiproliferative: Arbutus unedo (AuL), Cynara cardunculus (CyaA), Centaurea calcitrapa (CcA), Smilax aspera (SaA), and Tanacetum audibertii (TaA), the latter endemic to Sardinia and Corsica. Thus, their ability to induce cell cycle arrest and apoptosis was tested. All extracts determined cell cycle block in G2/M phase. Nevertheless, the p53 expression levels were increased only by TaA. The effector caspases were activated mainly by CycA, TaA, and CcA, while AuL and SaA did not induce apoptosis. The antiproliferative effects were also tested on human umbilical vein endothelial cells (HUVEC). Except for AuL, all the extracts were able to reduce significantly cell population, suggesting a potential antiangiogenic activity. The phytochemical composition was first explored by 1H NMR profiling, followed by further purifications to confirm the structure of the most abundant metabolites, such as phenolic compounds and sesquiterpene lactones, which might play a role in the measured bioactivity.

Correction to Chemical Fingerprint of Zn-Hydroxyapatite in the Early Stages of Osteogenic Differentiation.

[This corrects the article DOI: 10.1021/acscentsci.9b00509.].

Chemical Fingerprint of Zn-Hydroxyapatite in the Early Stages of Osteogenic Differentiation.

The core knowledge about biomineralization is provided by studies on the advanced phases of the process mainly occurring in the extracellular matrix. Here, we investigate the early stages of biomineralization by evaluating the chemical fingerprint of the initial mineral nuclei deposition in the intracellular milieu and their evolution toward hexagonal hydroxyapatite. The study is conducted on human bone mesenchymal stem cells exposed to an osteogenic cocktail for 4 and 10 days, exploiting laboratory X-ray diffraction techniques and cutting-edge developments of synchrotron-based 2D and 3D cryo-X-ray microscopy. We demonstrate that biomineralization starts with Zn-hydroxyapatite nucleation within the cell, rapidly evolving toward hexagonal hydroxyapatite crystals, very similar in composition and structure to the one present in human bone. These results provide experimental evidence of the germinal role of Zn in hydroxyapatite nucleation and foster further studies on the intracellular molecular mechanisms governing the initial phases of bone tissue formation.

Cytotoxic Effects of Artemisia annua L. and Pure Artemisinin on the D-17 Canine Osteosarcoma Cell Line.

Artemisia annua has been used for centuries in Traditional Chinese Medicine. Although used as an antimalarial drug, its active compound artemisinin and the semisynthetic derivatives have also been investigated for their anticancer properties, with interesting and promising results. The aims of this research were to evaluate (i) the cytotoxicity and the antiproliferative effect of pure artemisinin and a hydroalcoholic extract obtained from A. annua on the D-17 canine osteosarcoma cell line and (ii) the intracellular iron concentration and its correlation with the cytotoxic effects. Both artemisinin and hydroalcoholic extract induced a cytotoxic effect in a dose-dependent manner. Pure artemisinin caused an increase of cells in the S phase, whereas the hydroalcoholic extract induced an evident increase in the G2/M phase. A significant decrease of iron concentration was measured in D-17 cells treated with pure artemisinin and hydroalcoholic extract compared to untreated cells. In conclusion, although preliminary, the data obtained in this study are indicative of a more potent cytotoxic activity of the hydroalcoholic extract than pure artemisinin, indicating a possible synergistic effect of the phytocomplex and a mechanism of action involving iron and possibly ferroptosis. Considering the similarities between human and canine osteosarcomas, progress in deepening knowledge and improving therapeutic protocols will probably be relevant for both species, in a model of reciprocal translational medicine.

Analysis of Artemisia annua extracts and related products by high performance liquid chromatography-tandem mass spectrometry coupled to sample treatment miniaturisation.

Artemisinin, the main antimalarial compound of Artemisia annua L., is currently attracting increasing interest for its antiproliferative properties, but its content is highly variable, depending on several genetic, environmental and processing conditions. Aim of the present study is to analyse the artemisinin content in different plant extracts, to test their in vitro activity on cell proliferation and then to correlate these data to the active principle concentration. For this purpose, an innovative miniaturised sample pretreatment strategy based on microextraction by packed sorbent (MEPS) was developed and coupled to an original advanced method based on liquid chromatography with diode array detection and tandem mass spectrometry (LC-DAD-MS/MS). The method was fully validated, granting consistent data. Good linearity was found over a suitable concentration range, i.e. 5-1000ng/mL. Extraction yields (>85%), precision (RSD < 3.5%) and accuracy (recovery 88-93%) were all within acceptable levels of confidence. After validation, the method was successfully applied to the determination of artemisinin in A. annua extracts. Analyte content was widely variable (up to twenty-fold) according to the starting material and the extraction procedure, ranging between 5.9µg/g and 109µg/mL. The cytotoxic activity of all analysed extracts was also tested on human leukemic cells by viable cell count and cell cycle analysis. Artemisinin concentrations and biological activity were carefully evaluated and the observed antiproliferative effects varied according to artemisinin content in each extract type. This highlights the need to quantitatively analyse the main active constituent of plant extracts and the obtained data have shown to be promising for the choice of the related herbal product dosage.

Fluorescence lifetime imaging of intracellular magnesium content in live cells.

The first detailed analysis of FLIM applications for Mg cell imaging is presented. We employed the Mg-sensitive fluorescent dye named DCHQ5, a derivative of diaza-18-crown-6 ethers appended with two 8-hydroxyquinoline groups, to perform fluorescence lifetime imaging in control and Mg deprived SaOS-2 live cells, which contain different concentrations of magnesium. We found that the lifetime maps are almost uniform all over the cells and, most relevantly, we showed that the ratio of the amplitude terms is related to the magnesium intracellular concentration.

Magnesium Is a Key Regulator of the Balance between Osteoclast and Osteoblast Differentiation in the Presence of Vitamin D3.

Magnesium (Mg) is crucial for bone health. Low concentrations of Mg inhibit the activity of osteoblasts while promoting that of osteoclasts, with the final result of inducing osteopenia. Conversely, little is known about the effects of high concentrations of extracellular Mg on osteoclasts and osteoblasts. Since the differentiation and activation of these cells is coordinated by vitamin D3 (VD3), we investigated the effects of high extracellular Mg, as well as its impact on VD3 activity, in these cells. U937 cells were induced to osteoclastic differentiation by VD3 in the presence of supra-physiological concentrations (>1 mM) of extracellular Mg. The effect of high Mg concentrations was also studied in human bone-marrow-derived mesenchymal stem cells (bMSCs) induced to differentiate into osteoblasts by VD3. We demonstrate that high extra-cellular Mg levels potentiate VD3-induced osteoclastic differentiation, while decreasing osteoblastogenesis. We hypothesize that Mg might reprogram VD3 activity on bone remodeling, causing an unbalanced activation of osteoclasts and osteoblasts.